Peptides capable of binding to serum proteins and compounds, constructs and polypeptides comprising the same

ABSTRACT

The present invention relates to amino acid sequences that are capable of binding to serum proteins; to compounds, proteins, polypeptides, fusion proteins or constructs comprising or essentially consisting of such amino acid sequences; to nucleic acids that encode such amino acid sequences, compounds, proteins, polypeptides, fusion proteins or constructs; to compositions, and in particular pharmaceutical compositions, that comprise such amino acid sequences, compounds, proteins, polypeptides, fusion proteins or constructs; and to uses of such amino acid sequences, compounds, proteins, polypeptides, fusion proteins or constructs.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/424,986, filed Apr. 16, 2009, which claims the benefit under 35U.S.C. §119(e) of U.S. provisional application Ser. No. 61/045,690 filedon Apr. 17, 2008; of U.S. provisional application Ser. No. 61/050,385filed on May 5, 2008; and of U.S. provisional application Ser. No.61/119,803 filed on Dec. 4, 2008, the entire disclosures of which areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to amino acid sequences that are capableof binding to serum proteins; to peptides that comprise or essentiallyconsist of such amino acid sequences; to compounds and constructs (suchas fusion proteins and polypeptides) that comprise such amino acidsequences; to nucleic acids that encode such amino acid sequences,peptides, fusion proteins or polypeptides; to compositions, and inparticular pharmaceutical compositions, that comprise such amino acidsequences, peptides constructs, compounds, fusion proteins orpolypeptides; and to uses of such amino acid sequences, peptidesconstructs, compounds, fusion proteins or polypeptides.

Other aspects, embodiments, advantages and applications of the inventionwill become clear from the further description herein.

BACKGROUND OF THE INVENTION

The non-prepublished International application PCT/EP2007/063348entitled “Peptides capable of binding to serum proteins” describesmethods for generating peptides that are capable of binding to serumproteins, which peptides can be linked or fused to a therapeutic moiety,compound, protein or other therapeutic entity in order to increase thehalf-life thereof.

PCT/EP2007/063348 also describes a number of specific amino acidsequences that are capable of binding to human serum albumin and thatcan be linked or fused to a therapeutic moiety, compound, protein orother therapeutic entity in order to increase the half-life thereof.These amino acid sequences include the amino acid sequenceAASYSDYDVFGGGTDFGP (SEQ ID NO:1), which is called “17D12” inPCT/EP2007/063348 and which is listed in PCT/EP2007/063348 as SEQ IDNO:3.

It is an object of the present invention to provide amino acid sequenceswith improved binding to serum albumin, compared to the amino acidsequence AASYSDYDVFGGGTDFGP (SEQ ID NO:1). In particular, it is anobject of the invention to provide amino acid sequences that:

-   -   bind better (as defined herein) to human serum albumin than the        amino acid sequence AASYSDYDVFGGGTDFGP (SEQ ID NO:1),        and/or    -   can specifically bind (as defined herein) to human serum albumin        and that can also specifically bind serum albumin from at least        one other species of mammal (such as serum albumin from a mouse,        rat, rabbit, dog or a species of primate such as baboon or        rhesus monkey), and in particular can specifically bind to human        serum albumin and to serum albumin from cynomolgus monkey;        and/or    -   can bind to (human) serum albumin and that have other improved        properties for pharmaceutical use compared to the amino acid        sequence AASYSDYDVFGGGTDFGP (SEQ ID NO:1), such as improved        stability, improved protease resistance, etc.;        and/or    -   when linked or fused to a therapeutic moiety, compound, protein        or other therapeutic entity provide a greater increase of the        serum half-life or other pharmacologically relevant properties        than the amino acid sequence of SEQ ID NO:1 (when linked or        fused to the same therapeutic).

It is also an object of the invention to provide amino acid sequencesthat can be linked or fused to a therapeutic moiety, compound, proteinor other therapeutic entity, such that the resulting compound orconstruct has an improved half-life compared to a corresponding compoundor construct that contains the amino acid sequence AASYSDYDVFGGGTDFGP(SEQ ID NO:1).

SUMMARY OF THE INVENTION

It is an object of the present invention to provide amino acid sequencesthat are an alternative, and in particular an improved alternative, tothe serum protein-binding amino acid sequences described inPCT/EP2007/063348.

Generally, the invention achieves this objective by providing the aminoacid sequences described herein. These amino acid sequences can bind to(and in particular, specifically bind to, as defined herein) serumalbumin (and in particular to human serum albumin) and can be used assmall peptides or as peptide moieties for linking or fusing to atherapeutic compound (such as a therapeutic protein or polypeptide) inorder to increase the half-life thereof. These amino acid sequences(which are also referred to herein as “amino acid sequences of theinvention”) are as further defined herein.

Thus, according to a first aspect, the invention relates to an aminoacid sequence that:

-   a) has at least 50%, preferably at least 65%, more preferably at    least 70%, even more preferably at least 75%, such as at least 80%,    such as at least 90%, but not 100%, sequence identity (as defined    herein) with the amino acid sequence AASYSDYDVFGGGTDFGP (SEQ ID    NO:1);    and that:-   b) binds better to human serum albumin than the amino acid sequence    AASYSDYDVFGGGTDFGP (SEQ ID NO:1).

Another aspect of the invention relates to an amino acid sequence that:

-   a) that has no more than 9, preferably no more than 8, in particular    no more than 7, such as 6, 5, 4, 3, 2 or 1 amino acid difference(s)    (as defined herein) with the amino acid sequence AASYSDYDVFGGGTDFGP    (SEQ ID NO:1);    and that:-   b) binds better to human serum albumin than the amino acid sequence    AASYSDYDVFGGGTDFGP (SEQ ID NO:1).

In yet another aspect, the invention relates to an amino acid sequencethat

-   a) contains one or more of the following sequence motifs: DYDV (SEQ    ID NO:116), YDVF (SEQ ID NO:117), DVFG (SEQ ID NO:118), VFGG (SEQ ID    NO:119), FGGG (SEQ ID NO:120) and/or GGGT (SEQ ID NO:121);-   b) has a total length of between 5 and 50, preferably between 7 and    40, more preferably between 10 and 35, such as about 15, 20, 25 or    30 amino acid residues;    and that-   c) binds better to human serum albumin than the amino acid sequence    AASYSDYDVFGGGTDFGP (SEQ ID NO:1);    which amino acid sequence is not the sequence AASYSDYDVFGGGTDFGP    (SEQ ID NO:1).

In yet another aspect, the invention relates to an amino acid sequencethat

-   a) contains one or more of the following sequence motifs: DYDVF (SEQ    ID NO:122), YDVFG (SEQ ID NO:123), DVFGG (SEQ ID NO:124), VFGGG (SEQ    ID NO:125) and/or FGGGT (SEQ ID NO:126);-   b) has a total length of between 5 and 50, preferably between 7 and    40, more preferably between 10 and 35, such as about 15, 20, 25 or    30 amino acid residues;    and that-   c) binds better to human serum albumin than the amino acid sequence    AASYSDYDVFGGGTDFGP (SEQ ID NO:1);    which amino acid sequence is not the sequence AASYSDYDVFGGGTDFGP    (SEQ ID NO:1). Preferably, such an amino acid sequence is as further    described herein.

In yet another aspect, the invention relates to an amino acid sequencethat

-   a) contains one or more of the following sequence motifs: DYDVFG    (SEQ ID NO:127), YDVFGG (SEQ ID NO:128), DVFGGG (SEQ ID NO:129)    and/or VFGGGT (SEQ ID NO:130);-   b) has a total length of between 6 and 50, preferably between 7 and    40, more preferably between 10 and 35, such as about 15, 20, 25 or    30 amino acid residues;    and that-   c) binds better to human serum albumin than the amino acid sequence    AASYSDYDVFGGGTDFGP (SEQ ID NO:1);    which amino acid sequence is not the sequence AASYSDYDVFGGGTDFGP    (SEQ ID NO:1). Preferably, such an amino acid sequence is as further    described herein.

In yet another aspect, the invention relates to an amino acid sequencethat

-   a) contains one or more of the following sequence motifs: DYDVFGG    (SEQ ID NO:131), YDVFGGG (SEQ ID NO:132) and/or DVFGGGT (SEQ ID    NO:133);-   b) has a total length of between 7 and 50, preferably between 8 and    40, more preferably between 10 and 35, such as about 15, 20, 25 or    30 amino acid residues;    and that-   c) binds better to human serum albumin than the amino acid sequence    AASYSDYDVFGGGTDFGP (SEQ ID NO:1);    which amino acid sequence is not the sequence AASYSDYDVFGGGTDFGP    (SEQ ID NO:1). Preferably, such an amino acid sequence is as further    described herein.

In yet another aspect, the invention relates to an amino acid sequencethat

-   a) contains one or more of the following sequence motifs: DYDVFGGG    (SEQ ID NO:134) and/or YDVFGGGT (SEQ ID NO:135);-   b) has a total length of between 8 and 50, preferably between 9 and    40, more preferably between 10 and 35, such as about 15, 20, 25 or    30 amino acid residues;    and that-   c) binds better to human serum albumin than the amino acid sequence    AASYSDYDVFGGGTDFGP (SEQ ID NO:1);    which amino acid sequence is not the sequence AASYSDYDVFGGGTDFGP    (SEQ ID NO:1). Preferably, such an amino acid sequence is as further    described herein.

In yet another aspect, the invention relates to an amino acid sequencethat

-   a) contains the following sequence motif: DYDVFGGGT (SEQ ID NO:136);-   b) has a total length of between 9 and 50, preferably between 9 and    40, more preferably between 10 and 35, such as about 15, 20, 25 or    30 amino acid residues;    and that-   c) binds better to human serum albumin than the amino acid sequence    AASYSDYDVFGGGTDFGP (SEQ ID NO:1);    which amino acid sequence is not the sequence AASYSDYDVFGGGTDFGP    (SEQ ID NO:1). Preferably, such an amino acid sequence is as further    described herein.

The amino acid sequences of the invention (as further described herein)preferably (at least) contain:

-   (i) an Arg (R) residue, in particular an Arg (R) residue that is    capable of forming a hydrogen bond with the amino acid residues    Asn (N) 133 & Asn (N) 135 of human serum albumin and/or capable of    forming electrostatic interactions with the main-chain oxygen atoms    of the Pro (P) 134 and Leu (L) 136 residues of human serum albumin;    and/or-   (ii) a Trp (W) residue, in particular a Trp (W) residue that is    capable of forming electrostatic interactions with the Arg (R) 138    residue of human serum albumin; and/or-   (iii) the sequence motif GGG;    and preferably at least any two and more preferably all three of    (i), (ii) and (iii).

The amino acid sequences of the invention (as further described herein)preferably (at least) contain:

-   (i) the sequence motif RXWD, in which X may be any amino acid    sequence but is preferably W, Y, F, S or D; and/or-   (ii) the sequence motif GGG, preferably the sequence motif FGGG,    more preferably the sequence motif DVFGGG (SEQ ID NO:129), and in    particular the sequence motif DVFGGGT (SEQ ID NO:133);    and most preferably both these sequence motifs (i) and (ii).

Instead of the sequence motif DVFGGG (SEQ ID NO:129), an preferred aminoacid sequence of the invention may for example also contain the sequencemotif DAFGGG (SEQ ID NO:192). Also, instead of the sequence motifDVFGGGT (SEQ ID NO:133), a preferred amino acid sequence of theinvention may for example also contain the sequence motifs DVFGGGS (SEQID NO:193) or DAFGGGT (SEQ ID NO:194). Other similar sequence motifsthat may be present in the amino acid sequences of the invention will beclear to the skilled person based on the disclosure herein (such as thesequences mentioned in Table II and in Table V).

Thus, in another aspect, the invention relates to an amino acid sequencethat:

-   a) has at least 50%, preferably at least 65%, more preferably at    least 70%, even more preferably at least 75%, such as at least 80%,    such as at least 90%, but not 100%, sequence identity (as defined    herein) with the amino acid sequence AASYSDYDVFGGGTDFGP (SEQ ID    NO:1);    and that:-   b) binds better to human serum albumin than the amino acid sequence    AASYSDYDVFGGGTDFGP (SEQ ID NO:1);    and that:-   c) comprises an Arg (R) residue, in particular an Arg (R) residue    that is capable of forming a hydrogen bond with the amino acid    residues Asn (N) 133 & Asn (N) 135 of human serum albumin and/or    capable of forming electrostatic interactions with the main-chain    oxygen atoms of the Pro (P) 134 and Leu (L) 136 residues of human    serum albumin.

This amino acid sequence preferably also comprises (i) the sequencemotif RXWD, in which X may be any amino acid sequence but is preferablyW, Y, F, S or D; and/or (ii) the sequence motif GGG, preferably thesequence motif FGGG, more preferably the sequence motif DVFGGG (SEQ IDNO:129), and in particular the sequence motif DVFGGGT (SEQ ID NO:133);and most preferably both these sequence motifs.

The above amino acid sequence is also preferably as further describedherein.

In another aspect, the invention relates to an amino acid sequence that:

-   a) has at least 50%, preferably at least 65%, more preferably at    least 70%, even more preferably at least 75%, such as at least 80%,    such as at least 90%, but not 100%, sequence identity (as defined    herein) with the amino acid sequence AASYSDYDVFGGGTDFGP (SEQ ID    NO:1);    and that:-   b) binds better to human serum albumin than the amino acid sequence    AASYSDYDVFGGGTDFGP (SEQ ID NO:1);    and that:-   c) comprises a Trp (W) residue, in particular a Trp (W) residue that    is capable of forming electrostatic interactions with the Arg (R)    138 residue of human serum albumin.

This amino acid sequence preferably also comprises (i) the sequencemotif RXWD, in which X may be any amino acid sequence but is preferablyW, Y, F, S or D; and/or (ii) the sequence motif GGG, preferably thesequence motif FGGG, more preferably the sequence motif DVFGGG (SEQ IDNO:129), and in particular the sequence motif DVFGGGT (SEQ ID NO:133);and most preferably both these sequence motifs.

The above amino acid sequence is also preferably as further describedherein.

In another aspect, the invention relates to an amino acid sequence that:

-   a) has at least 50%, preferably at least 65%, more preferably at    least 70%, even more preferably at least 75%, such as at least 80%,    such as at least 90%, but not 100%, sequence identity (as defined    herein) with the amino acid sequence AASYSDYDVFGGGTDFGP (SEQ ID    NO:1);    and that:-   b) binds better to human serum albumin than the amino acid sequence    AASYSDYDVFGGGTDFGP (SEQ ID NO:1);    and that:-   c) comprises an Arg (R) residue, in particular an Arg (R) residue    that is capable of forming a hydrogen bond with the amino acid    residues Asn (N) 133 & Asn (N) 135 of human serum albumin and/or    capable of forming electrostatic interactions with the main-chain    oxygen atoms of the Pro (P) 134 and Leu (L) 136 residues of human    serum albumin;    and that-   d) comprises a Trp (W) residue, in particular a Trp (W) residue that    is capable of forming electrostatic interactions with the Arg (R)    138 residue of human serum albumin.

This amino acid sequence preferably also comprises (i) the sequencemotif RXWD, in which X may be any amino acid sequence but is preferablyW, Y, F, S or D; and/or (ii) the sequence motif GGG, preferably thesequence motif FGGG, more preferably the sequence motif DVFGGG (SEQ IDNO:129), and in particular the sequence motif DVFGGGT (SEQ ID NO:133);and most preferably both these sequence motifs.

The above amino acid sequences are also preferably as further describedherein.

Some preferred, but non-limiting sequence motifs that may be present inthe amino acid sequences of the invention are:

-   -   the amino acid sequence RXWDXDVFGGG (SEQ ID NO: 171), in which        the first (from the N-terminal end) amino acid residue indicated        by X is chosen from Y, S or D; and the second amino acid residue        indicated by X is chosen from Y or F.    -   the amino acid sequence RXWDXDVFGGGT (SEQ ID NO: 172), in which        the first (from the N-terminal end) amino acid residue indicated        by X is chosen from Y, S or D; and the second amino acid residue        indicated by X is chosen from Y or F.    -   the amino acid sequence RXWDXDVFGGGTP (SEQ ID NO: 173), in which        the first (from the N-terminal end) amino acid residue indicated        by X is chosen from Y, S or D; and the second amino acid residue        indicated by X is chosen from Y or F.    -   the amino acid sequence RXWDXDVFGGGTPG (SEQ ID NO: 174), in        which the first (from the N-terminal end) amino acid residue        indicated by X is chosen from Y, S or D; and the second amino        acid residue indicated by X is chosen from Y or F.    -   the amino acid sequence RXWDXDVFGGGTPGG (SEQ ID NO: 175), in        which the first (from the N-terminal end) amino acid residue        indicated by X is chosen from Y, S or D; and the second amino        acid residue indicated by X is chosen from Y or F.    -   an amino acid sequence chosen from RYWDYDVFGGG (SEQ ID NO: 176);        RDWDFDVFGGG (SEQ ID NO: 177); RSWDFDVFGGG (SEQ ID NO: 178) or        RYWDFDVFGGG (SEQ ID NO: 179); and in particular chosen from        RDWDFDVFGGG (SEQ ID NO: 177); RSWDFDVFGGG (SEQ ID NO: 178) or        RYWDFDVFGGG (SEQ ID NO: 179).    -   an amino acid sequence chosen from RYWDYDVFGGGT (SEQ ID NO:        180); RDWDFDVFGGGT (SEQ ID NO: 181); RSWDFDVFGGGT (SEQ ID        NO: 182) or RYWDFDVFGGGT (SEQ ID NO: 183); and in particular        chosen from RDWDFDVFGGGT (SEQ ID NO: 181); RSWDFDVFGGGT (SEQ ID        NO: 182) or RYWDFDVFGGGT (SEQ ID NO: 183).    -   an amino acid sequence chosen from RYWDYDVFGGGTP (SEQ ID NO:        184); RDWDFDVFGGGTP (SEQ ID NO: 185); RSWDFDVFGGGTP (SEQ ID        NO: 186) or RYWDFDVFGGGTP (SEQ ID NO: 187); and in particular        chosen from RDWDFDVFGGGTP (SEQ ID NO: 185); RSWDFDVFGGGTP (SEQ        ID NO: 186) or RYWDFDVFGGGTP (SEQ ID NO: 187)    -   an amino acid sequence chosen from RYWDYDVFGGGTPV (SEQ ID NO:        188); RDWDFDVFGGGTPV (SEQ ID NO: 189); RSWDFDVFGGGTPV (SEQ ID        NO: 190) or RYWDFDVFGGGTPV (SEQ ID NO: 191); and in particular        chosen from RDWDFDVFGGGTPV (SEQ ID NO: 189); RSWDFDVFGGGTPV (SEQ        ID NO: 190) or RYWDFDVFGGGTPV (SEQ ID NO: 191).

In the context of the present invention, an amino acid sequence of theinvention is deemed to “bind better” to serum albumin (such as humanserum albumin or serum albumin from another species of mammal, such asserum albumin of cynomolgus monkey) than the amino acid sequence of SEQID NO:1:

-   -   when it binds to said serum albumin with a higher specificity        than the amino acid sequence of SEQ ID NO:1; and/or    -   when it binds to said serum albumin with a higher affinity (as        defined herein, and expressed as a K_(D), K_(A), k_(on) or        k_(off) rate, and determined using one of the methods described        herein) than the amino acid sequence of SEQ ID NO:1; and/or    -   when it binds stronger to said serum albumin than the amino acid        sequence of SEQ ID NO:1 in the phage-ELISA assay described in        Example 2 below; and/or    -   when it binds better to said serum albumin than the amino acid        sequence of SEQ ID NO:1 in the solution binding competition        ELISA described in Example 3 below;    -   when it binds better to said serum albumin than the amino acid        sequence of SEQ ID NO:1 in the solution binding competition        ELISA described in Example 5 below;    -   when it binds to said serum albumin with a higher avidity (i.e.        when the amino acid sequence of the invention is used as a        concatamer) than the amino acid sequence of SEQ ID NO:1 (i.e.        when it is used in the form of a comparable concatamer); and/or    -   when a compound of the invention (as defined herein) that        comprises one or more of said amino acid sequences of the        invention binds to said serum albumin with a higher specificity,        affinity and/or avidity than a corresponding compound of the        invention that comprises one or more amino acid sequences of SEQ        ID NO:1 (for example as determined using the BIAcore™        measurement used in Example 6). For example, and without        limitation, an amino acid sequence of the invention is said to        bind better to serum albumin when a fusion protein in which the        relevant amino acid sequence is fused (optionally via a suitable        linker) to the Nanobody 2D3 (SEQ ID NO: 137) binds with a higher        specificity, affinity and/or avidity to serum albumin than a        corresponding fusion protein in which the Nanobody 2D3 is fused        (optionally via the same suitable linker) to the amino acid        sequence AASYSDYDVFGGGTDFGP (SEQ ID NO:1) (for example as        determined using the BIAcore™ measurement used in Example 6).        For the purposes of this comparison, the relevant amino acid        sequence and amino acid sequence AASYSDYDVFGGGTDFGP (SEQ ID        NO:1) may for example (but without limitation) be linked to the        C-terminus of 2D3 (optionally via the same suitable linker). As        a specific but non-limiting example thereof, the specificity,        affinity and/or avidity for serum albumin of a fusion protein        that corresponds to the 2D3-56H5 fusion protein given in SEQ ID        NO: 139 (in which the amino acid sequence 56H5 has been replaced        by the relevant amino acid sequence) may be compared to the        specificity, affinity and/or avidity for serum albumin of the        corresponding fusion protein 2D3-17D12 given in SEQ ID NO: 138        (for example as determined using the BIAcore™ measurement used        in Example 6).

In particular, “binding” as described herein may be determined using thesolution binding competition assay described in Example 3 or Example 9;or, when the amino acid sequences is expressed as a fusion with theNanobody 2D3 as described in Example 7 or 10, in the Biacore assaysdescribed in these Examples.

Preferably, the amino acid sequences of the invention are such that theybind equally well or preferably better to human serum albumin than theamino acid sequence 56E4 of the invention (SEQ ID NO:14). For thispurpose, such an amino acid sequence of the invention may for example bethe amino acid sequence 56E4 of the invention (SEQ ID NO:14) or anvariant of the amino acid sequence 56E4 that is such that it bindsequally well or preferably better to human serum albumin than the aminoacid sequence 56E4, such as an affinity matured version of the aminoacid sequence 56E4. Some preferred, but non-limiting examples of suchamino acid sequences of the invention are given in Example 9 and TableV, and comprise the amino acid sequences 59E4 (SEQ ID NO:14); 59A5 (SEQID NO: 147); 59C8 (SEQ ID NO: 148); 59F2 (SEQ ID NO: 149); 59B3 (SEQ IDNO: 150); 59B2 (SEQ ID NO: 151); 60E6 (SEQ ID NO: 152); 60F1 (SEQ ID NO:153); 60G5 (SEQ ID NO: 154); 59H12 (SEQ ID NO: 155); 59C2 (SEQ ID NO:156); and 59H10 (SEQ ID NO: 157); of which 59F2 (SEQ ID NO: 149); 59C2(SEQ ID NO: 156) and 59H12 (SEQ ID NO: 155) are particularly preferred.

Thus, in another aspect, the invention relates to an amino acid sequencethat

-   a) is the sequence AARYWDYDVFGGGTPVGG (56E4; SEQ ID NO:14); or-   b) has at least 65%, more preferably at least 70%, even more    preferably at least 75%, such as at least 80%, for example at least    85% or at least 90% with the amino acid sequence AARYWDYDVFGGGTPVGG    (56E4; SEQ ID NO:14); and/or-   c) that has no more than 6, preferably no more than 5, in particular    no more than 4, such as 3, 2 or 1 amino acid difference(s) (as    defined herein) with the amino acid sequence AARYWDYDVFGGGTPVGG    (56E4; SEQ ID NO:14)    and that preferably:-   d) binds equally well and preferably better to human serum albumin    than the amino acid sequence AARYWDYDVFGGGTPVGG (56E4; SEQ ID    NO:14).

Again, such amino acid sequences are incorporated into the meaning ofthe term “amino acid sequences of the invention” as used in its broadestsense herein; and they are preferably as further described herein. Thus,for example, such amino acid sequences preferably comprise (i) an Arg(R) residue, in particular an Arg (R) residue that is capable of forminga hydrogen bond with the amino acid residues Asn (N) 133 & Asn (N) 135of human serum albumin and/or capable of forming electrostaticinteractions with the main-chain oxygen atoms of the Pro (P) 134 and Leu(L) 136 residues of human serum albumin; and/or (ii) a Trp (W) residue,in particular a Trp (W) residue that is capable of forming electrostaticinteractions with the Arg (R) 138 residue of human serum albumin; and/or(iii) the sequence motif GGG; and preferably at least any two and morepreferably all three of (i), (ii) and (iii). In particular, such aminoacid sequences of the invention preferably (at least) contain (i) thesequence motif RXWD, in which X may be any amino acid sequence but ispreferably W, Y, F, S or D; and/or (ii) the sequence motif GGG,preferably the sequence motif FGGG, more preferably the sequence motifDVFGGG (SEQ ID NO:129), and in particular the sequence motif DVFGGGT(SEQ ID NO:133); and most preferably both these sequence motifs (i) and(ii).

In another aspect, the invention relates to an amino acid sequence that

-   a) is one of the amino acid sequences 59A5 (SEQ ID NO: 147); 59C8    (SEQ ID NO: 148); 59F2 (SEQ ID NO: 149); 59B3 (SEQ ID NO: 150); 59B2    (SEQ ID NO: 151); 60E6 (SEQ ID NO: 152); 60F1 (SEQ ID NO: 153); 60G5    (SEQ ID NO: 154); 59H12 (SEQ ID NO: 155); 59C2 (SEQ ID NO: 156); or    59H10 (SEQ ID NO: 157); or-   b) has at least 65%, more preferably at least 70%, even more    preferably at least 75%, such as at least 80%, for example at least    85% or at least 90% with at least one of the amino acid sequences    59A5 (SEQ ID NO: 147); 59C8 (SEQ ID NO: 148); 59F2 (SEQ ID NO: 149);    59B3 (SEQ ID NO: 150); 59B2 (SEQ ID NO: 151); 60E6 (SEQ ID NO: 152);    60F1 (SEQ ID NO: 153); 60G5 (SEQ ID NO: 154); 59H12 (SEQ ID NO:    155); 59C2 (SEQ ID NO: 156); and/or 59H10 (SEQ ID NO: 157); and/or-   c) that has no more than 6, preferably no more than 5, in particular    no more than 4, such as 3, 2 or 1 amino acid difference(s) (as    defined herein) with at least one of the amino acid sequences 59A5    (SEQ ID NO: 147); 59C8 (SEQ ID NO: 148); 59F2 (SEQ ID NO: 149); 59B3    (SEQ ID NO: 150); 59B2 (SEQ ID NO: 151); 60E6 (SEQ ID NO: 152); 60F1    (SEQ ID NO: 153); 60G5 (SEQ ID NO: 154); 59H12 (SEQ ID NO: 155);    59C2 (SEQ ID NO: 156); and/or 59H10 (SEQ ID NO: 157);    and that preferably:-   d) binds equally well and preferably better to human serum albumin    than the amino acid sequence AARYWDYDVFGGGTPVGG (56E4; SEQ ID    NO:14).

Again, such amino acid sequences are incorporated into the meaning ofthe term “amino acid sequences of the invention” as used in its broadestsense herein; and they are preferably as further described herein.

In another aspect, the invention relates to an amino acid sequence that

-   a) is one of the amino acid sequences 59F2 (SEQ ID NO: 149); 59H12    (SEQ ID NO: 155); or 59C2 (SEQ ID NO: 156); or-   b) has at least 65%, more preferably at least 70%, even more    preferably at least 75%, such as at least 80%, for example at least    85% or at least 90% with at least one of the amino acid sequences    59F2 (SEQ ID NO: 149); 59H12 (SEQ ID NO: 155); and/or 59C2 (SEQ ID    NO: 156); and/or-   c) that has no more than 6, preferably no more than 5, in particular    no more than 4, such as 3, 2 or 1 amino acid difference(s) (as    defined herein) with at least one of the amino acid sequences 59F2    (SEQ ID NO: 149); 59H12 (SEQ ID NO: 155); and/or 59C2 (SEQ ID NO:    156);    and that preferably:-   d) binds equally well and preferably better to human serum albumin    than the amino acid sequence AARYWDYDVFGGGTPVGG (56E4; SEQ ID    NO:14).

Again, such amino acid sequences are incorporated into the meaning ofthe term “amino acid sequences of the invention” as used in its broadestsense herein; and they are preferably as further described herein.

According to another aspect, the invention relates to an amino acidsequence that has at least 50%, preferably at least 65%, more preferablyat least 70%, even more preferably at least 75%, such as at least 80%,such as at least 90%, but not 100%, sequence identity (as definedherein) with the amino acid sequence AASYSDYDVFGGGTDFGP (SEQ ID NO:1);wherein said amino acid sequence is such that, when it is linked orfused to a therapeutic moiety, compound, protein or other therapeuticentity, the compound of the invention (as defined herein) thus obtainedhas a longer half-life (as defined herein) than a corresponding compoundor construct in which said therapeutic moiety, compound, protein orother therapeutic entity is linked or fused to the amino acid sequenceof SEQ ID NO:1 (i.e. instead of the amino acid sequence of theinvention).

According to yet another aspect, the invention relates to an amino acidsequence that has no more than 9, preferably no more than 8, inparticular no more than 7, such as 6, 5, 4, 3, 2 or 1 amino aciddifference(s) (as defined herein) with the amino acid sequenceAASYSDYDVFGGGTDFGP (SEQ ID NO:1); wherein said amino acid sequence issuch that, when it is linked or fused to a therapeutic moiety, compound,protein or other therapeutic entity, the compound of the invention (asdefined herein) thus obtained has a longer half-life (as defined herein)than a corresponding compound or construct in which said therapeuticmoiety, compound, protein or other therapeutic entity is linked or fusedto the amino acid sequence SEQ ID NO:1 (i.e. instead of the amino acidsequence of the invention).

Other aspects, embodiments, uses and advantages of the present inventionwill become clear from the further description herein.

Some representative, but non-limiting examples of amino acid sequencesof the invention are listed as SEQ ID NO's: 2 to 115 in Table II and inSEQ ID NO's: 147 to 157 in Table V below (with some preferredrepresentative examples being marked in bold typeface and underlined).

Some particularly preferred representative examples of amino acidsequences of the invention are the amino acid sequences PMP56G11 (SEQ IDNO:68); PMP56E4 (SEQ ID NO: 14); PMP54H4 (SEQ ID NO: 106); PMP54H5 (SEQID NO: 33); PMP56H1 (SEQ ID NO: 31); PMP56E2 (SEQ ID NO:47); PMP56G3(SEQ ID NO: 35); PMP54G1 (SEQ ID NO:38); PMP56F1 (SEQ ID NO: 30);PMP54H2 (SEQ ID NO: 40); PMP56H9 (SEQ ID NO: 100); PMP56F2 (SEQ ID NO:51); PMP26A3 (SEQ ID NO:26) and 01B3 (SEQ ID NO:115); and in particular59E4 (SEQ ID NO:14); 59A5 (SEQ ID NO: 147); 59C8 (SEQ ID NO: 148); 59F2(SEQ ID NO: 149); 59B3 (SEQ ID NO: 150); 59B2 (SEQ ID NO: 151); 60E6(SEQ ID NO: 152); 60F1 (SEQ ID NO: 153); 60G5 (SEQ ID NO: 154); 59H12(SEQ ID NO: 155); 59C2 (SEQ ID NO: 156); and 59H10 (SEQ ID NO: 157); ofwhich 59F2 (SEQ ID NO: 149); 59C2 (SEQ ID NO: 156) and 59H12 (SEQ ID NO:155) are particularly preferred.

Generally, the amino acid sequences of the invention will contain(within the overall limitations set out herein) one or more “amino aciddifferences” (as defined herein) with the amino acid sequenceAASYSDYDVFGGGTDFGP (SEQ ID NO:1), such that the resulting amino acidsequence of the invention binds better (as defined herein) to humanserum albumin than the amino acid sequence AASYSDYDVFGGGTDFGP (SEQ IDNO:1).

Generally, and within the overall limitations set out herein, such anamino acid difference may comprise an insertion, deletion orsubstitution or one or more amino acid residues at one or morepositions, compared to the sequence of SEQ ID NO:1. Usually, compared tothe sequence of SEQ ID NO:1, an amino acid sequence of the inventioncontains at least one amino acid substitution (such as those mentionedherein); and optionally also one or more amino acid insertions and/orone or more amino acid deletions.

Suitable substitutions, insertions and/or deletions (and combinationsthereof) will be clear to the skilled person based on the disclosureherein, and for example include one or more of the substitutions,insertions and/or deletions that are present in the amino acid sequencesof SEQ ID NOs: 2 to 115 and in SEQ ID NO's: 147 to 157 (and inparticular in the amino acid sequences that are preferred among theamino acid sequences of SEQ ID NOs: 2 to 115 and/or and in the aminoacid sequences of SEQ ID NO's: 147 to 157), or any suitable combinationof these substitutions, insertions and/or deletions. For this purpose,an alignment of the sequence of SEQ ID NO:1 and the sequences of SEQ IDNOs: 2 to 115 are given in FIG. 1) and in Table V, the sequences of SEQID NO's: 147 to 157 are compared to the sequence of SEQ ID NO: 14.

Some preferred, but non-limiting, examples of possible substitutionsthat can be present in an amino acid sequence of the invention (comparedto the amino acid sequence of SEQ ID NO:1) are listed in Table I below(it being understood that an amino acid sequence of the invention can,within the limits set out herein, contain one or more further suitableamino acid substitutions, insertions or deletions).

It should be noted that in the most preferred amino acid sequences ofthe invention, position 3 is most preferably R, position 5 is W(preferably in combination with a D on position 6); position 7 ispreferably F (but may also be Y or W); position 15 is P and position 16is V.

By comparison, in the sequence of SEQ ID NO:1, position 3 is S; position5 is S; position 7 is Y; position 15 is D, position 15 is D; andposition 16 is F.

The most preferred amino acid sequences of the invention share thefollowing residues with the sequence of SEQ ID NO:1: the Y at position 4(although, in the sequences of the invention, this may also be F, W, Sor D); the D at position 6; the DVFGGG (SEQ ID NO:129) motif atpositions 8-13 (although this may also be DAFGGG (SEQ ID NO:192) in thepreferred sequences of the invention), and the T at position 14; as wellas the G at position 17.

TABLE I Examples of possible substitutions that can be present in anamino acid sequence of the invention. position in a.a. in SEQ ID NO: SEQID Examples of possible substitutions in an 143 and Position NO: 1 aminoacid sequence of the invention Example 8 1 A A (preferred) or V 2 2 A A(preferred), G or V 3 3 S R (preferred), L, F, Y, W, P, T, S, M, 4 A, D,I, K, Q or V; 4 Y Y, F, W, S or D 5 5 S Y, R, W, F, L, D, P, G, H, K, M,S, T; 6 of which W is much preferred in combination with a D on position6 7 Y Y, F or W; of which an F preferred 8 11 G G (preferred) or A 12 15D P (preferred), A, D, S, V, E, G, Q, R, 16 W or Y 16 F V, L, E, G, S,R, K, A, P, Q, D, M, F, 17 I, T 18 P G, E, A, V, S, D, T, N, I, Q, R orW 19

Optionally, based on the disclosure herein (such as Table II below), theskilled person will also be able to determine other (or additional)suitable substitutions, insertions and/or deletions (or combinationsthereof) by means of limited trail-and-error, for example by testing acandidate amino acid sequence that comprises the intended substitutions,insertions and/or deletions for binding to human serum albumin, forexample using the assay of Example 2 and/or Example 3 below (in whichsaid candidate amino acid sequence may then optionally be compared tothe amino acid sequence of SEQ ID NO:1 and/or to one or more of theamino acid sequences of SEQ ID NOs: 2 to 115 and/or SEQ ID NO's: 147 to157).

Again, such amino acid sequences are preferably as further describedherein. Thus, for example, such amino acid sequences preferably comprise(i) an Arg (R) residue, in particular an Arg (R) residue that is capableof forming a hydrogen bond with the amino acid residues Asn (N) 133 &Asn (N) 135 of human serum albumin and/or capable of formingelectrostatic interactions with the main-chain oxygen atoms of the Pro(P) 134 and Leu (L) 136 residues of human serum albumin; and/or (ii) aTrp (W) residue, in particular a Trp (W) residue that is capable offorming electrostatic interactions with the Arg (R) 138 residue of humanserum albumin; and/or (iii) the sequence motif GGG; and preferably atleast any two and more preferably all three of (i), (ii) and (iii). Inparticular, such amino acid sequences of preferably (at least) contain(i) the sequence motif RXWD, in which X may be any amino acid sequencebut is preferably W, Y, F, S or D; and/or (ii) the sequence motif GGG,preferably the sequence motif FGGG, more preferably the sequence motifDVFGGG (SEQ ID NO:129), and in particular the sequence motif DVFGGGT(SEQ ID NO:133); and most preferably both these sequence motifs (i) and(ii).

Generally, when an amino acid sequence of the invention contains one ormore amino acid substitutions compared to the amino acid sequenceAASYSDYDVFGGGTDFGP (SEQ ID NO:1), these may be conservative amino acidsubstitutions (as defined herein) or non-conservative amino acidsubstitutions (it being understood by the skilled person that suitablenon-conservative amino acid substitutions will generally be more likelyto improve, or further improve, the binding to human serum albumin).

Other amino acid sequences of the invention may be provided byintroducing suitable amino acid substitutions, insertions and/ordeletions (or combinations thereof) in one of the amino acid sequencesof SEQ ID NOs: 2 to 115 and/or SEQ ID NO's: 147 to 157, such that theresulting amino acid sequence of the invention binds better (as definedherein) to human serum albumin than the amino acid sequenceAASYSDYDVFGGGTDFGP (SEQ ID NO:1). Again, these may be conservative aminoacid substitutions (as defined herein) or non-conservative amino acidsubstitutions (it being understood by the skilled person that suitableconservative amino acid substitutions will generally be more likely toensure that the favourable binding to human serum albumin is retained,or even improved).

From the disclosure herein, it will be clear that the amino acidsequences of the invention preferably either contain, compared to thesequence of SEQ ID NO:1, no amino acid substitutions or deletions (andpreferably also no insertions) at the positions 4, 6, 7, 8, 9, 10, 12,13, 14 or 17; or only a limited number (i.e. 3, 2 or preferably only 1)amino acid substitutions or deletions compared to the sequence of SEQ IDNO:1 (which then preferably are conservative substitutions as definedherein). The reason for this is that, from the alanine scanningexperiment described in Example 4, it has become clear that introducingamino acid substitutions or deletions, although not excluded from thescope of the invention, may carry an increased risk of reducing thebinding to human serum albumin.

In another preferred, but non-limiting aspect, the amino acid sequencesof the invention preferably contain a least one proline residue, such as1, 2, 3 or 4 proline residues. In particular, the amino acid sequencesof the invention may contain (a) proline residue(s) at one or more (suchas any one, two, three or four) of the positions 1, 2, 3, 5, 11, 15, 16or 18 (and in particular 3, 5, 15, 16 and/or 18). Proline residues mayalso be inserted next to or near these positions.

According to one preferred, but non-limiting aspect, an amino acidsequence of the invention may comprise one or more (such as any two, anythree, any four or all five) of the following amino acid substitutionscompared to the amino acid sequence of SEQ ID NO.1:

-   -   the serine residue (S) at position 3 of SEQ ID NO:1 is replaced        by an amino acid residue chosen from arginine (R), proline (P),        an aromatic amino acid residue (F, Y, W or H; in particular F, Y        or W) or a hydrophobic amino acid residue (L, I, V or M);        and/or    -   the serine residue (S) at position 5 of SEQ ID NO:1 is replaced        by an amino acid residue chosen from arginine (R), proline (P),        or an aromatic amino acid residue (F, Y, W or H; in particular        F, Y or W);        and/or    -   the aspartate residue (D) at position 15 of SEQ ID NO:1 is        replaced by an amino acid residue chosen from proline (P) or a        small amino acid residue (A, G, S or T);        and/or    -   the phenylalanine residue (F) at position 16 of SEQ ID NO:1 is        replaced by proline (P), a hydrophobic amino acid residue (L, I,        V or M), or a or a small amino acid residue (A, G, S or T);        and/or    -   the proline residue (P) at position 18 of SEQ ID NO:1 is        maintained or replaced by a (partially) negative amino acid        residue (D, E, Q or N) or a small amino acid residue (A, G, S or        T);        and optionally one or more further suitable amino acid        insertions, deletions and/or substitutions (as further described        herein).

In a particularly preferred subclass of amino acid sequences of theinvention, the serine residue (S) at position 3 of SEQ ID NO:1 isreplaced by arginine (R). These amino acid sequences may comprise one ormore further amino acid insertions, deletions and/or substitutions asdescribed herein.

In particular, in amino acid sequences of the invention with an R atposition 3:

-   -   the serine residue (S) at position 5 of SEQ ID NO:1 is replaced        by an amino acid residue chosen from proline (P) or an aromatic        amino acid residue (F, Y, W or H; in particular F, Y or W);        and/or    -   the aspartate residue (D) at position 15 of SEQ ID NO:1 is        replaced by an amino acid residue chosen from proline (P) or a        small amino acid residue (A, G, S or T);        and/or    -   the phenylalanine residue (F) at position 16 of SEQ ID NO:1 is        replaced by proline (P), a hydrophobic amino acid residue (L, I,        V or M), or a or a small amino acid residue (A, G, S or T);        and/or    -   the proline residue (P) at position 18 of SEQ ID NO:1 is        maintained or replaced by a (partially) negative amino acid        residue (D, E, Q or N) or a small amino acid residue (A, G, S or        T);        and optionally one or more further suitable amino acid        insertions, deletions and/or substitutions (as further described        herein).

Some preferred amino acid sequences within the amino acid sequences ofthe invention are the amino acid sequences of SEQ ID NO: 2 to 115 and/orSEQ ID NO's: 147 to 157, or amino acid sequences that have not more than3, such as 3, 2, or 1 amino acid differences with one of the amino acidsequences of SEQ ID NO: 2 to 115 and/or SEQ ID NO's: 147 to 157 (inwhich said amino acid differences are preferably as generally describedherein for the amino acid sequences of the invention).

Some more preferred amino acid sequences within the amino acid sequencesof the invention are the amino acid sequences of SEQ ID NOs: 5, 7, 9,14, 25, 26, 30, 31, 33, 34, 35, 36, 38, 40, 42, 47, 51, 55, 66, 68, 86,94, 97, 100, 103, 106, 111; 115 and in particular 147, 148, 149, 150,151, 152, 153, 154, 155, 156 and/or 147; or amino acid sequences thathave not more than 3, such as 3, 2, or 1 amino acid differences with oneof these amino acid sequences (in which said amino acid differences arepreferably as generally described herein for the amino acid sequences ofthe invention).

Some particularly preferred amino acid sequences within the amino acidsequences of the invention are the amino acid sequences PMP56G11 (SEQ IDNO:68); PMP56E4 (SEQ ID NO: 14); PMP54H4 (SEQ ID NO: 106); PMP54H5 (SEQID NO: 33); PMP56H1 (SEQ ID NO: 31); PMP56E2 (SEQ ID NO:47); PMP56G3(SEQ ID NO: 35); PMP54G1 (SEQ ID NO:38); PMP56F1 (SEQ ID NO: 30);PMP54H2 (SEQ ID NO: 40); PMP56H9 (SEQ ID NO: 100); PMP56F2 (SEQ ID NO:51); PMP26A3 (SEQ ID NO:26) or 01B3 (SEQ ID NO:115); and in particular59E4 (SEQ ID NO:14); 59A5 (SEQ ID NO: 147); 59C8 (SEQ ID NO: 148); 59F2(SEQ ID NO: 149); 59B3 (SEQ ID NO: 150); 59B2 (SEQ ID NO: 151); 60E6(SEQ ID NO: 152); 60F1 (SEQ ID NO: 153); 60G5 (SEQ ID NO: 154); 59H12(SEQ ID NO: 155); 59C2 (SEQ ID NO: 156); and 59H10 (SEQ ID NO: 157) (ofwhich 59F2 (SEQ ID NO: 149); 59C2 (SEQ ID NO: 156) and 59H12 (SEQ ID NO:155) are particularly preferred); or amino acid sequences that have notmore than 3, such as 3, 2, or 1 amino acid differences with one of theseamino acid sequences (in which said amino acid differences arepreferably as generally described herein for the amino acid sequences ofthe invention).

Preferably, an amino acid sequence of the invention has a total size ofbetween 9 and 27 amino acid residues, such as between 12 and 24 aminoacid residues, for example between 15 and 21 amino acid residues, suchas 16, 17, 18, 19 or 20 amino acid residues).

The amino acid sequences of the invention can also be provided and/orused in the form of a peptide in which the amino acid sequence is linkedto a small flanking sequence (e.g. of no more than 10, preferably of nomore than 5 amino acid residues) at the C-terminus, the N-terminus, orboth. These may for example be present because the amino acid sequenceof the invention (or a compound of the invention in which said aminoacid sequence is present) has been obtained by expression of acorresponding nucleotide sequence, in which the nucleotide sequence thatencodes the amino acid sequence of the invention is either preceded by(i.e. at the 5′-end) and/or followed by (i.e. at the 3′-end) by a smallnucleotide sequence that encodes a restriction site or that forms partof a cloning site (and that leads to the presence of the flankingsequence(s) in the expressed peptide). Examples of such flankingsequences are the amino acid sequences GSA and AAA.

The amino acid sequences described herein can bind to serum albumin in a“non-constrained” format (i.e. not comprising any disulphide bridges),and can advantageously be used in such a non-constrained format. It ishowever included in the scope of the invention that the amino acidsequences described herein are provided in, and/or are used in, a“constrained” format, for example in the form of a peptide in which anamino acid sequence of the invention is flanked by two flankingsequences that can form a disulphide bridge between them (for a furtherdescription hereof, reference is made to PCT/EP2007/063348).

The amino acid sequence of the invention is preferably such that itbinds to serum albumin (and in particular to human serum albumin) insuch a way that the half-life of the serum albumin molecule is not(significantly) reduced.

Preferably, the amino acid sequence of the invention binds to serumalbumin or at least one part, fragment, epitope or domain thereof; andin particular to human serum albumin or at least one part, fragment,epitope or domain thereof. When the amino acid sequence of the inventionbinds to (human) serum albumin, it preferably is capable of binding toamino acid residues on serum albumin that are not involved in binding of(human) serum albumin to FcRn; and/or of binding to amino acid residueson serum albumin that do not form part of domain III of (human) serumalbumin Reference is made to WO 06/0122787.

Generally, the amino acid sequences of the invention are such that theybind better to human serum albumin than the amino acid sequence of SEQID NO:1. Preferably, the amino acid sequences of the invention are suchthat they bind equally well or better to human serum albumin than theamino acid sequence of SEQ ID NO:14. As mentioned, “binding” asdescribed herein may in particular be determined using the solutionbinding competition assay described in Example 3 or Example 9; or, whenthe amino acid sequences is expressed as a fusion with the Nanobody 2D3as described in Example 7 or 10, in the Biacore assays described inthese Examples.

Preferably, any amino acid sequence of the invention as described hereinhas a total length of between 5 and 50, preferably between 7 and 40,more preferably between 10 and 35, such as about 15, 20, 25 or 30 aminoacid residues.

Also, preferably, amino acid sequences of the invention are such that,when they are linked or fused to a therapeutic moiety, compound, proteinor other therapeutic entity, the compound of the invention (as definedherein) thus obtained has a longer half-life (as defined herein) than acorresponding compound or construct in which said therapeutic moiety,compound, protein or other therapeutic entity is linked or fused to theamino acid sequence of SEQ ID NO:1 (i.e. instead of the amino acidsequence of the invention). This may in particular be determined byfusing the amino acid sequence of the invention to the Nanobody 2D3 inthe manner described in Example 6 or Example 10, and then by determiningthe pharmacokinetic profile as described in Example 7 or Example 13.

In particular, in a preferred aspect, the amino acid sequences of theinvention are such that, when they are linked or fused to a therapeuticmoiety, compound, protein or other therapeutic entity, the compound ofthe invention (as defined herein) thus obtained has a similar or longerhalf-life (as defined herein) than a corresponding compound or constructin which said therapeutic moiety, compound, protein or other therapeuticentity is linked or fused to the amino acid sequence of SEQ ID NO:14(56E4).

The amino acid sequences of the invention are preferably alsocross-reactive (as defined herein) with the serum albumin from at leastone species of mammal other than man; an in particular cross-reactivewith serum albumin from cynomolgus monkey.

Generally, the amino acid sequences of the invention are also preferablysuch that they compete with the peptide of SEQ ID NO:1 and/or with thepeptide of SEQ ID NO:14 for binding to human serum albumin, and/or suchthat they cross-block (as defined herein) the binding of the peptide ofSEQ ID NO:1 and/or the binding of the peptide of SEQ ID NO:14 to humanserum albumin.

The amino acid sequences of the invention are preferably such that theycan bind to one or more of the following amino acid residues of humanserum albumin (numbering as indicated in Example 8): Asn (N) 133; Pro(P) 134; Asn (N) 135; Leu (L) 136; Leu (L) 139; Arg (R) 141; Tyr (Y)162; Glu (E) 165; He (I) 166; His (H) 170; Phe (F) 173; Phe (F) 181; Gly(G) 213; Lys (K) 214; Ser (S) 217; Gln (Q) 483; and/or Lys (K) 543;and/or such that they can compete with the amino acid sequence of SEQ IDNO:1 and/or the amino acid sequence of SEQ ID NO:14 for binding to oneor more of these amino acid residues; and/or such that they cancross-block the binding of the amino acid sequence of SEQ ID NO:1 and/orthe binding of the amino acid sequence of SEQ ID NO:14 to one or more ofthese amino acid residues.

More in particular, the amino acid sequences of the invention arepreferably such that they can bind to an epitope on human serum albuminthat comprises either (i) the stretch of amino acid residues thatcomprises the residues Asn (N) 133; Pro (P) 134; Asn (N) 135; Leu (L)136; Leu (L) 139 and Arg (R) 141; and/or (ii) the stretch of amino acidresidues that comprises the residues Tyr (Y) 162; Glu (E) 165; Ile (I)166; His (H) 170; Phe (F) 173; Phe (F) 181; and/or (iii) the stretch ofamino acid residues that comprises the residues Gly (G) 213; Lys (K) 214and Ser (S) 217; and/or such that they can compete with the amino acidsequence of SEQ ID NO:1 and/or the amino acid sequence of SEQ ID NO:14for binding to one of these stretches of amino acid residues; and/orsuch that they can cross-block the binding of the amino acid sequence ofSEQ ID NO:1 and/or the binding of the amino acid sequence of SEQ IDNO:14 to one or more of these stretches of amino acid residues.

Even more in particular; the amino acid sequences of the invention arepreferably such that they can bind to a hydrophobic subpocket on humanserum albumin that is comprises (amongst others) residues the residuesLeu (L) 139, Glu (E) 165, Ile (I) 166, His (H) 170, Phe (F) 173, Phe (F)181, Gly (G) 213, Lys (K) 214, Ser (S) 217 and Gln (Q) 483; and/or suchthat they can compete with the amino acid sequence of SEQ ID NO:1 and/orthe amino acid sequence of SEQ ID NO:14 for binding to this subpocket;and/or such that they can cross-block the binding of the amino acidsequence of SEQ ID NO:1 and/or the binding of the amino acid sequence ofSEQ ID NO:14 to this subpocket.

The above peptides may be as further described herein; and may forexample be affinity matured variants of the peptide of SEQ ID NO:1, andmay in particular be affinity matured variants of the peptide of SEQ IDNO: 14.

In one specific aspect, the invention does not comprise the amino acidsequences that are mentioned in FIG. 4 or FIG. 8 of PCT/EP2007/063348.

The amino acid sequences of the invention (or a compound of theinvention comprising at least one such amino acid sequence, as furtherdescribed herein) are preferably such that they can bind to a serumalbumin, and in particular to human serum albumin:

-   -   with a dissociation constant (K_(D)) in the range of 10⁻⁵ to        10⁻¹² moles/liter or less, and preferably in the range of 10⁻⁷        to 10⁻¹² moles/liter or less and more preferably in the range of        10⁻⁸ to 10⁻¹² moles/liter (i.e. with an association constant        (K_(A)) of in the range of 10⁵ to 10¹² liter/moles or more, and        preferably in the range of 10⁷ to 10¹² liter/moles or more, and        more preferably in the range of 10⁸ to 10¹² liter/moles), such        that said dissociation constant is better (i.e. smaller/lower)        than the dissociation constant with which the amino acid        sequence AASYSDYDVFGGGTDFGP (SEQ ID NO:1) binds to human serum        albumin;        and/or    -   with a k_(on)-rate in the range of between 10² M⁻¹s⁻¹ to about        10⁷ M⁻¹s⁻¹, preferably in the range between 10² M⁻¹s⁻¹ and 10⁷        M⁻¹s⁻¹, more preferably in the range between 10⁴ M⁻¹s⁻¹ and 10⁷        M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such that        said k_(on)-rate is better (i.e. higher) than the k_(on)-rate        with which the amino acid sequence AASYSDYDVFGGGTDFGP (SEQ ID        NO:1) binds to human serum albumin;        and/or    -   with a k_(off) rate in the range between 1 s⁻¹ (t_(1/2)=0.69 s)        and 10⁻⁶ s⁻¹ (providing a near irreversible complex with a        t_(1/2) of multiple days), preferably in the range between 10⁻²        s⁻¹ and 10⁻⁶ s⁻¹, more preferably in the range between 10⁻³ s⁻¹        and 10⁻⁴ s⁻¹, such as in the range between 10⁻⁴ s⁻¹ and 10⁻⁶        s⁻¹, such that said k_(off)-rate is better (i.e. higher) than        the k_(off)-rate with which the amino acid sequence        AASYSDYDVFGGGTDFGP (SEQ ID NO:1) binds to human serum albumin.

Preferably, an amino acid sequence of the invention (or a compound ofthe invention comprising one such amino acid sequence, as furtherdescribed herein) is such that it will bind to human serum albumin withan affinity less than 1000 nM, preferably less than 500 nM, preferablyless than 200 nM, more preferably less than 10 nM, such as less than 500pM; such that said affinity is better (i.e. smaller/lower) than theaffinity with which the amino acid sequence AASYSDYDVFGGGTDFGP (SEQ IDNO:1) binds to human serum albumin.

The amino acid sequences of the invention (as well as compounds of theinvention comprising the same, as defined herein) are preferably suchthat they bind to or otherwise associate with human serum albumin insuch a way that, when the amino acid sequence (or compound) is bound toor otherwise associated with a human serum albumin in man, it exhibits aserum half-life of at least about 50% (such as about 50% to 70%),preferably at least 60% (such as about 60% to 80%), or preferably atleast 70% (such as about 70% to 90%), more preferably at least 80% (suchas about 80% to 90%), or preferably at least about 90% of the naturalhalf-life of the human serum albumin in man.

The amino acid sequences of the invention may bind to serum albumin(such as human serum albumin) in a conditional manner (as described inthe International application PCT/EP2007/060850 of Ablynx N.V.), i.e.such that:

-   a) they bind to human serum albumin molecule under a first    biological condition with a dissociation constant (K_(D)) of 10⁻⁵    moles/liter or less; and-   b) they bind to human serum albumin under a second biological    condition with a dissociation constant (K_(D)) that is at least 10    fold different from (and in particular more than) the dissociation    constant with which said amino acid sequence binds to said desired    molecule under said first biological condition.

in which the first and second biological conditions may be as describedin the International application PCT/EP2007/060850 of Ablynx N.V. Inparticular, as described in the International applicationPCT/EP2007/060850, the first biological condition and the secondbiological condition may differ in respect of pH, in which said firstbiological condition may comprise a physiological pH of more than 7.0,for example a pH of more than 7.1 or a pH of more than 7.2, such as a pHin the range of 7.2 to 7.4; and the second biological condition maycomprise a physiological pH of less than 7.0, for example a pH of lessthan 6.7 or a pH of less than 6.5, such as a pH in the range of 6.5 to6.0 (or visa versa).

Preferably, however, amino acid sequences of the invention may bind toserum albumin (such as human serum albumin) in a manner that is“essentially independent of the pH” (as described in the Internationalapplication PCT/EP2007/060849 of Ablynx N.V., and as further definedherein).

In one non-limiting aspect, the amino acid sequences of the inventionare preferably cross-reactive (as defined herein) with serum albuminfrom at least one other species of mammal, for example from mouse,rabbit, rat, or a primate. In particular, the amino acid sequences ofthe invention may be cross-reactive with serum albumin from a primatechosen from the group consisting of monkeys from the genus Macaca (suchas, and in particular, cynomolgus monkeys (Macaca fascicularis) and/orrhesus monkeys (Macaca mulatta) and baboon (Papio ursinus), andpreferably at least with cyno serum albumin Also, when an amino acidsequence of the invention is cross-reactive with serum albumin from sucha species of primate, it is preferably such that, when it is bound to orassociated with a serum albumin molecule in said primate, it exhibits aserum half-life of at least about 50% (such as about 50% to 70%),preferably at least about 60% (such as about 60% to 80%), or preferablyat least about 70% (such as about 70% to 90%), more preferably at leastabout 80% (such as about 80% to 90%), or preferably at least about 90%of the natural half-life of said serum albumin in said primate.

The invention also relates to a compound or construct which comprises atleast one amino acid sequence of the invention and at least onetherapeutic moiety (also referred to herein as “compounds of theinvention”). These compounds or constructs may be as further describedherein, and may for example be polypeptide or protein constructs thatcomprise or essentially consist of at least one amino acid sequence ofthe invention that is linked to at least one therapeutic moiety,optionally via one or more suitable linkers or spacers. Such polypeptideor protein constructs may for example (without limitation) be a fusionprotein, as further described herein.

Such compounds of the invention may contain one, two, three or moreamino acid sequences of the invention, suitably linked to the at leastone therapeutic moiety (and optionally to each other), optionally viaone or more suitable linkers (as described herein). Also, when acompound of the invention comprises two, three or more amino acidsequences of the invention, these may be the same or different.

In one specific aspect, such compounds of the invention may comprise oneamino acid sequence of the invention, suitably linked to the at leastone therapeutic moiety, optionally via one or more suitable linkers (asdescribed herein). For example, in such a case, when the therapeuticmoiety is a protein or polypeptide (such that the resulting compound ofthe invention is a fusion protein), the amino acid sequence of theinvention may either be linked to the C-terminus of the therapeuticmoiety or to the N-terminus of the therapeutic moiety (again, optionallyvia a suitable linker).

In another specific aspect, such compounds of the invention may comprisetwo amino acid sequence of the invention, suitably linked to the atleast one therapeutic moiety (and optionally to each other), optionallyvia one or more suitable linkers (as described herein).

More specifically, such compounds of the invention may comprise twoamino acid sequence of the invention, that are each suitably linked tothe at least one therapeutic moiety (i.e. on different attachment sitesof the therapeutic moiety), again optionally via suitable linkers. Forexample, in such a case, when the therapeutic moiety is a protein orpolypeptide (such that the resulting compound of the invention is afusion protein), one amino acid sequence of the invention may forexample be linked to the C-terminus of the therapeutic moiety (again,optionally via a suitable linker) and one amino acid sequence of theinvention may for example be linked to the N-terminus of the therapeuticmoiety (again, optionally via a suitable linker).

Alternatively, such compounds of the invention may comprise two (ormore) amino acid sequences of the invention that are linked to eachother (again, optionally via a suitable linker) so as to form a “tandemrepeat”, which tandem repeat may then be suitably linked to the at leastone therapeutic moiety (again optionally via a suitable linker). Forexample, in such a case, when the therapeutic moiety is a protein orpolypeptide (such that the resulting compound of the invention is afusion protein), the tandem repeat of the two or more amino acidsequences of the invention may either be linked to the C-terminus of thetherapeutic moiety or to the N-terminus of the therapeutic moiety(again, optionally via a suitable linker).

Other suitable combinations of two or more amino acid sequences of theinvention and one or more therapeutic moieties (again, optionally linkedvia suitable linkers) will be clear to the skilled person based on thedisclosure herein.

In another aspect, the compounds of the invention comprise two or more(such as two, three or four) therapeutic moieties (which may be the sameor different), and one or more (such as two, three, four or more) aminoacid sequences of the invention (which may also be the same ordifferent), in which the two or more (such as two, three or four)therapeutic moieties and/or the one or more (such as two, three, four ormore) amino acid sequences of the invention may be suitably linked toeach other (again optionally via one or more suitable linkers) so as toform a compound of the invention. For example, in such compounds of theinvention, the two or more therapeutic moieties may be suitably linkedto each other (again optionally via one or more suitable linkers), andone or more of the amino acid sequences of the invention (and/or one ormore tandem repeats of two or more amino acid sequences of theinvention, as described herein) may be linked (again, optionally via oneor more suitable linkers) to any (or all) of the therapeutic moieties.

Also, in a further aspect, one or more of the linker(s) used to link thetwo or more therapeutic moieties to each other may comprise one or moreof the amino acid sequences of the invention, and such linkerscomprising one or more amino acid sequences of the invention (optionallycomprising one or more further linking amino acid sequences to link theacid sequences of the invention to each other and/or to one or moretherapeutic moieties) form a further aspect of the invention.

For example, when a compound of the invention comprises two therapeuticmoieties (which may be the same or different), some examples of possiblebut non-limiting configurations of the above compounds of the inventionare:

[TM]-[L]-[AA]-[L]-[TM]

[AA]-[L]-[TM]-[L]-[TM]

[TM]-[L]-[TM]-[L]-[AA]

[TM]-[L]-[AA]-[L]-[AA]-[TM]

[AA]-[L]-[TM]-[L]-[TM]-[L]-[AA]

[AA]-[L]-[AA]-[TM]-[L]-[TM]

[TM]-[L]-[TM]-[L]-[AA]-[AA]

[AA]-[L]-[TM]-[L]-[AA]-[L]-[TM]-[L]-[AA]

[AA]-[L]-[TM]-[L]-[AA]-[L]-[AA]-[L]-[TM]-[L]-[AA]

in which “[TM]” refers to the therapeutic moiety, “[L]” refers to alinker (which in each case is optional), and “[AA]” refers to an aminoacid sequence of the invention. Other suitable configuration will beclear to the skilled person based on the disclosure herein. Again, inthese constructs, when there are two or more linkers and/or amino acidsequences of the invention present, these may be the same or different.Again, when the therapeutic moieties and the linkers are proteins or(polypeptides), the above constructs may be fusion proteins or fusionconstructs (which may for example be suitably obtained by suitableexpression of a corresponding nucleic acid or nucleotide sequence).

In another aspect, the invention relates to a polypeptide construct thatcomprises two or more (and in particular two or three, and preferablytwo) amino acid sequences of the invention, in which the two or moreamino acid sequences of the invention present in said polypeptide may bethe same or different; and in which the two or more amino acid sequencesof the invention may be either linked directly to each other, or linkedto each other via a suitable linker (as further described herein). Sucha “tandem repeat” construct of the invention may again be linked to oneor more therapeutic moieties, in the same way as a single amino acidsequence of the invention. In some cases, the use of a tandem repeat mayprovide for an (even further) improved affinity to human serum albumin(compared to the use of a single amino acid sequence of the invention)and/or for an (even further) improved half-life for the compounds of theinvention that contain such a tandem repeat (compared to a compound ofthe invention that comprises a single amino acid sequence of theinvention). A non-limiting example of the use of such a tandem repeatand of a compound of the invention that comprises such a tandem repeatis given in Example 14. Also, as described herein, such a tandem repeatconstruct may be used as a linker.

Such tandem repeats preferably contain two or more of the preferredamino acid sequences of the invention (which may be the same ordifferent), and in particular the particularly preferred amino acidsequences of the invention, such as (for example) 56E4 and affinitymatured variants of 56E4 such as 59H12, 59F2 and/or 59C2, all asdescribed herein. The invention also relates to compounds and constructsthat comprise such tandem repeats (which may again be fusion proteins);to nucleotide sequences or nucleic acids encoding such tandem repeats ofsuch fusion proteins, and to uses of such tandem repeats (e.g. to extendhalf-life and/or as linkers).

Thus, in another aspect, the invention relates to a polypeptideconstruct that comprises two or more (and in particular two or three,and preferably two) amino acid sequences of the invention, in which thetwo or more amino acid sequences of the invention present in saidpolypeptide may be the same or different; and in which the two or moreamino acid sequences of the invention may be either linked directly toeach other, or linked to each other via a suitable linker (as furtherdescribed herein); and in which each amino acid sequence presenttherein:

-   a) is one of the amino acid sequences 59A5 (SEQ ID NO: 147); 59C8    (SEQ ID NO: 148); 59F2 (SEQ ID NO: 149); 59B3 (SEQ ID NO: 150); 59B2    (SEQ ID NO: 151); 60E6 (SEQ ID NO: 152); 60F1 (SEQ ID NO: 153); 60G5    (SEQ ID NO: 154); 59H12 (SEQ ID NO: 155); 59C2 (SEQ ID NO: 156); or    59H10 (SEQ ID NO: 157); or-   b) has at least 65%, more preferably at least 70%, even more    preferably at least 75%, such as at least 80%, for example at least    85% or at least 90% with at least one of the amino acid sequences    59A5 (SEQ ID NO: 147); 59C8 (SEQ ID NO: 148); 59F2 (SEQ ID NO: 149);    59B3 (SEQ ID NO: 150); 59B2 (SEQ ID NO: 151); 60E6 (SEQ ID NO: 152);    60F1 (SEQ ID NO: 153); 60G5 (SEQ ID NO: 154); 59H12 (SEQ ID NO:    155); 59C2 (SEQ ID NO: 156); and/or 59H10 (SEQ ID NO: 157); and/or-   c) has no more than 6, preferably no more than 5, in particular no    more than 4, such as 3, 2 or 1 amino acid difference(s) (as defined    herein) with at least one of the amino acid sequences 59A5 (SEQ ID    NO: 147); 59C8 (SEQ ID NO: 148); 59F2 (SEQ ID NO: 149); 59B3 (SEQ ID    NO: 150); 59B2 (SEQ ID NO: 151); 60E6 (SEQ ID NO: 152); 60F1 (SEQ ID    NO: 153); 60G5 (SEQ ID NO: 154); 59H12 (SEQ ID NO: 155); 59C2 (SEQ    ID NO: 156); and/or 59H10 (SEQ ID NO: 157);    and preferably:-   d) binds equally well and preferably better to human serum albumin    than the amino acid sequence AARYWDYDVFGGGTPVGG (56E4; SEQ ID    NO:14).

Again, the amino acid sequences present in such a tandem repeat may beas further described herein, and the tandem repeat may be linked to oneor more therapeutic moieties, in the manner described herein.

Thus, in another aspect, the invention relates to a polypeptideconstruct that comprises two or more (and in particular two or three,and preferably two) amino acid sequences of the invention, in which thetwo or more amino acid sequences of the invention present in saidpolypeptide may be the same or different; and in which the two or moreamino acid sequences of the invention may be either linked directly toeach other, or linked to each other via a suitable linker (as furtherdescribed herein); and in which each amino acid sequence presenttherein:

-   a) is one of the amino acid sequences 59F2 (SEQ ID NO: 149); 59H12    (SEQ ID NO: 155); or 59C2 (SEQ ID NO: 156); or-   b) has at least 65%, more preferably at least 70%, even more    preferably at least 75%, such as at least 80%, for example at least    85% or at least 90% with at least one of the amino acid sequences    59F2 (SEQ ID NO: 149); 59H12 (SEQ ID NO: 155); and/or 59C2 (SEQ ID    NO: 156); and/or-   c) has no more than 6, preferably no more than 5, in particular no    more than 4, such as 3, 2 or 1 amino acid difference(s) (as defined    herein) with at least one of the amino acid sequences 59F2 (SEQ ID    NO: 149); 59H12 (SEQ ID NO: 155); and/or 59C2 (SEQ ID NO: 156);    and preferably:-   d) binds equally well and preferably better to human serum albumin    than the amino acid sequence AARYWDYDVFGGGTPVGG (56E4; SEQ ID    NO:14).

Again, the amino acid sequences present in such a tandem repeat may beas further described herein, and the tandem repeat may be linked to oneor more therapeutic moieties, in the manner described herein.

The at least one therapeutic moiety present in the compounds of theinvention preferably comprises or essentially consists of an amino acidsequence, and may in particular comprise or essentially consist of animmunoglobulin sequence or an antigen-binding fragment thereof (forexample, an antibody or an antigen-binding fragment thereof), such as animmunoglobulin variable domain or an antigen-binding fragment thereof(for example, a V_(H)-domain, a V_(L)-domain, a V_(HH)-domain or anantigen-binding fragment thereof); or a protein or polypeptidecomprising the same (for example, an scFv construct). For suchconstructs, reference is for example made to the review by Holliger andHudson, Nat. Biotechnol. 2005 September; 23(9):1126-36 and the furtherprior art cited therein.

According to one specific, but non-limiting aspect, the therapeuticmoiety comprises or essentially consists of a (single) domain antibody,a “dAb”, or a Nanobody®.

When the one or more therapeutic moieties are directed against one ormore pharmaceutically relevant targets, they may be directed against anysuitable target known per se. For example, when the therapeutic moietycomprises or essentially consists of a (single) domain antibody, a“dAb”, or a Nanobody®, it may for example be a dAb or Nanobody,IGN-gamma (see for example WO 04/041863), IgE (see for example WO04/041867), EGFR (see for example WO 05/044858; WO 07/066,106 or WO07/080,392); vWF (see for example WO 04/062551 or WO 06/1222825); IGF-IR(see for example WO 07/042,289); IL-6 (see for example WO 07/110,219);IL-6R (see for example WO 08/020,079); GPCR's (see for example WO08/074,839); chemokines (see for example WO 08/077,945); VEGF or itsreceptors (see for example WO 07/080,392; WO 08/101,985; WO 08/149,147;WO 08/149,146; or WO 08/149,150); RANK-L (see for example WO08/142,164); IL-R1 (see for example WO 06/059108; WO 07/063,311; WO07/063,308; or WO 08/149,149); TNF-R1 (see for example WO o6/038027; WO07/049,017; WO 08/149,148 or WO 08/149,144); IL-4 or IL-13 (see forexample WO 07/085,815); CD40L (see for example WO 06/030220).

The therapeutic moieties may also be other proteins or peptides with aknown therapeutical and/or pharmacological actions, such as, for exampleand without limitation, GLP-1; insulin; EPO; somatropin; interferons,interleukins and (other) cytokines and/or protein drugs used in cancertherapy.

In a compound of the invention the one or more amino acid sequences ofthe invention may be either directly linked to the at least onetherapeutic moiety or linked to the at least one therapeutic moiety viaone or more suitable linkers or spacers. Suitable linkers will be clearto the skilled person, for example based on the further disclosureherein. Some preferred, but non-limiting linkers are those mentioned onpages 127 and 128 of the International application WO 08/020,079 ofAblynx N.V., and include the “gly-ser linkers” mentioned therein.

When the one or more therapeutic moieties are amino acid sequences, thelinkers or spacers preferably comprise or essentially consist of aminoacid sequences, so that the resulting compound or construct essentiallyconsists of a (fusion) protein or (fusion) polypeptide (also referred toherein as a “polypeptide of the invention”).

In a further aspect, the invention relates to a compound of theinvention (as further defined herein) that comprises at least one aminoacid sequence that has at least 50%, preferably at least 60%, morepreferably at least 70%, even more preferably at least 75%, such as atleast 80%, at least 85%, at least 90% or at least 95%, but not 100%,sequence identity (as defined herein) with the amino acid sequenceAASYSDYDVFGGGTDFGP (SEQ ID NO:1), wherein said compound of the inventionhas a longer half-life (as defined herein) than a corresponding compoundthat, instead of said amino acid sequence(s), contains the amino acidsequence AASYSDYDVFGGGTDFGP (SEQ ID NO:1). Preferably, such a compoundhas a half-life that is essentially the same or longer than acorresponding compound that, instead of said amino acid sequence(s),contains the amino acid sequence 56E4 (SEQ ID NO:14). Again, the aminoacid sequence(s) present in such a compound may be as further describedherein; and are preferably amino acid sequences of the invention thatare described herein as being preferred.

In a further aspect, the invention relates to a compound of theinvention (as further defined herein) that comprises at least one aminoacid sequence that that has no more than 10, preferably no more than 9,more preferably no more than 8, even more preferably no more than 7,such as 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein)with the amino acid sequence AASYSDYDVFGGGTDFGP (SEQ ID NO:1), whereinsaid compound of the invention has a longer half-life (as definedherein) than a corresponding compound that, instead of said amino acidsequence(s), contains the amino acid sequence AASYSDYDVFGGGTDFGP (SEQ IDNO:1). Preferably, such a compound has a half-life that is essentiallythe same or longer than a corresponding compound that, instead of saidamino acid sequence(s), contains the amino acid sequence 56E4 (SEQ IDNO:14). Again, the amino acid sequence(s) present in such a compound maybe as further described herein; and are preferably amino acid sequencesof the invention that are described herein as being preferred.

In a further aspect, the invention relates to a compound of theinvention that comprises at least two amino acid sequences of theinvention. In another aspect, the invention relates to a compound of theinvention that comprises at least one tandem repeat (as defined herein)of at least two amino acid sequences of the invention. Preferably, saidcompound of the invention has a longer half-life (as defined herein)than a corresponding compound that, instead of said amino acidsequences, contains the same number of copies of the amino acid sequenceAASYSDYDVFGGGTDFGP (SEQ ID NO:1). More preferably, such a compound has ahalf-life that is essentially the same or longer than a correspondingcompound that, instead of said amino acid sequence(s), contains the samenumber of copies of the amino acid sequence 56E4 (SEQ ID NO:14). Again,the amino acid sequence(s) present in such a compound may be as furtherdescribed herein; and are preferably amino acid sequences of theinvention that are described herein as being preferred.

Some other aspects of the invention relate to the following peptides.Again, such peptides are incorporated into the meaning of the term“amino acid sequences of the invention” as used in its broadest senseherein; and these peptides are preferably as further described hereinfor the amino acid sequences of the invention.

Thus, in another aspect, the invention relates to a peptide that isspecific for (as defined herein) for human serum albumin and that bindsbetter (as defined herein) to HSA than the amino acid sequence 56E4 (SEQID NO: 14).

In another aspect, the invention relates to a peptide that is specificfor (as defined herein) for human serum albumin and that is an affinitymatured variant of the amino acid sequence 56E4 (SEQ ID NO:14)

In another aspect, the invention relates to a peptide that is specificfor (as defined herein) for human serum albumin and that comprises anArg (R) residue; and the sequence motif DVFGGG (SEQ ID NO:129), inparticular the sequence motif DVFGGGT (SEQ ID NO:133).

In another aspect, the invention relates to a peptide that is specificfor (as defined herein) for human serum albumin and that comprises anArg (R) residue that is capable of forming a hydrogen bond with theamino acid residues Asn (N) 133 & Asn (N) 135 of human serum albuminand/or capable of forming electrostatic interactions with the main-chainoxygen atoms of the Pro (P) 134 and Leu (L) 136 residues of human serumalbumin; and the sequence motif DVFGGG (SEQ ID NO:129), in particularthe sequence motif DVFGGGT (SEQ ID NO:133).

In another aspect, the invention relates to a peptide that is specificfor (as defined herein) for human serum albumin and that comprises a Trp(W) residue; and the sequence motif DVFGGG (SEQ ID NO:129), inparticular the sequence motif DVFGGGT (SEQ ID NO:133).

In another aspect, the invention relates to a peptide that is specificfor (as defined herein) for human serum albumin and that comprises a Trp(W) residue that is capable of forming electrostatic interactions withthe Arg (R) 138 residue of human serum albumin; and the sequence motifDVFGGG (SEQ ID NO:129), in particular the sequence motif DVFGGGT (SEQ IDNO:133).

In another aspect, the invention relates to a peptide that is specificfor (as defined herein) for human serum albumin and that comprises anArg (R) residue; a Trp (W) residue; and the sequence motif DVFGGG (SEQID NO:129), in particular the sequence motif DVFGGGT (SEQ ID NO:133).

In another aspect, the invention relates to a peptide that is specificfor (as defined herein) for human serum albumin and that comprises anArg (R) residue; an aromatic amino acid residue that is capable offorming electrostatic interactions with the Arg (R) 138 residue of humanserum albumin; and the sequence motif DVFGGG (SEQ ID NO:129), inparticular the sequence motif DVFGGGT (SEQ ID NO:133).

In another aspect, the invention relates to a peptide that is specificfor (as defined herein) for human serum albumin and that comprises anArg (R) residue that is capable of forming a hydrogen bond with theamino acid residues Asn (N) 133 & Asn (N) 135 of human serum albuminand/or capable of forming electrostatic interactions with the main-chainoxygen atoms of the Pro (P) 134 and Leu (L) 136 residues of human serumalbumin; a Trp (W) residue that is capable of forming electrostaticinteractions with the Arg (R) 138 residue of human serum albumin; andthe sequence motif DVFGGG (SEQ ID NO:129), in particular the sequencemotif DVFGGGT (SEQ ID NO:133).

In another aspect, the invention relates to a peptide that is specificfor (as defined herein) for human serum albumin and that comprises theamino acid sequence RXWDXDVFGGG (SEQ ID NO: 171), in which the first(from the N-terminal end) amino acid residue indicated by X is chosenfrom Y, S or D; and the second amino acid residue indicated by X ischosen from Y or F.

In another aspect, the invention relates to a peptide that is specificfor (as defined herein) for human serum albumin and that comprises theamino acid sequence RXWDXDVFGGGT (SEQ ID NO: 172), in which the first(from the N-terminal end) amino acid residue indicated by X is chosenfrom Y, S or D; and the second amino acid residue indicated by X ischosen from Y or F.

In another aspect, the invention relates to a peptide that is specificfor (as defined herein) for human serum albumin and that comprises theamino acid sequence RXWDXDVFGGGTP (SEQ ID NO: 173), in which the first(from the N-terminal end) amino acid residue indicated by X is chosenfrom Y, S or D; and the second amino acid residue indicated by X ischosen from Y or F.

In another aspect, the invention relates to a peptide that is specificfor (as defined herein) for human serum albumin and that comprises theamino acid sequence RXWDXDVFGGGTPG (SEQ ID NO: 174), in which the first(from the N-terminal end) amino acid residue indicated by X is chosenfrom Y, S or D; and the second amino acid residue indicated by X ischosen from Y or F.

In another aspect, the invention relates to a peptide that is specificfor (as defined herein) for human serum albumin and that comprises theamino acid sequence RXWDXDVFGGGTPGG (SEQ ID NO: 175), in which the first(from the N-terminal end) amino acid residue indicated by X is chosenfrom Y, S or D; and the second amino acid residue indicated by X ischosen from Y or F.

In another aspect, the invention relates to a peptide that is specificfor (as defined herein) for human serum albumin and that comprises anamino acid sequence chosen from RYWDYDVFGGG (SEQ ID NO: 176);RDWDFDVFGGG (SEQ ID NO: 177); RSWDFDVFGGG (SEQ ID NO: 178) orRYWDFDVFGGG (SEQ ID NO: 179); and in particular chosen from RDWDFDVFGGG(SEQ ID NO: 177); RSWDFDVFGGG (SEQ ID NO: 178) or RYWDFDVFGGG (SEQ IDNO: 179).

In another aspect, the invention relates to a peptide that is specificfor (as defined herein) for human serum albumin and that comprises anamino acid sequence chosen from RYWDYDVFGGGT (SEQ ID NO: 180);RDWDFDVFGGGT (SEQ ID NO: 181); RSWDFDVFGGGT (SEQ ID NO: 182) orRYWDFDVFGGGT (SEQ ID NO: 183); and in particular chosen fromRDWDFDVFGGGT (SEQ ID NO: 181); RSWDFDVFGGGT (SEQ ID NO: 182) orRYWDFDVFGGGT (SEQ ID NO: 183).

In another aspect, the invention relates to a peptide that is specificfor (as defined herein) for human serum albumin and that comprises anamino acid sequence chosen from RYWDYDVFGGGTP (SEQ ID NO: 184);RDWDFDVFGGGTP (SEQ ID NO: 185); RSWDFDVFGGGTP (SEQ ID NO: 186) orRYWDFDVFGGGTP (SEQ ID NO: 187); and in particular chosen fromRDWDFDVFGGGTP (SEQ ID NO: 185); RSWDFDVFGGGTP (SEQ ID NO: 186) orRYWDFDVFGGGTP (SEQ ID NO: 187).

In another aspect, the invention relates to a peptide that is specificfor (as defined herein) for human serum albumin and that comprises anamino acid sequence chosen from RYWDYDVFGGGTPV (SEQ ID NO: 188);RDWDFDVFGGGTPV (SEQ ID NO: 189); RSWDFDVFGGGTPV (SEQ ID NO: 190) orRYWDFDVFGGGTPV (SEQ ID NO: 191); and in particular chosen fromRDWDFDVFGGGTPV (SEQ ID NO: 189); RSWDFDVFGGGTPV (SEQ ID NO: 190) orRYWDFDVFGGGTPV (SEQ ID NO: 191).

In another aspect, the invention relates to a peptide that is specificfor (as defined herein) for human serum albumin and that comprises thesequence motif RXWD (in which X is chosen from W, Y, F, S or D) and thesequence motif FGGG.

In another aspect, the invention relates to a peptide that is specificfor (as defined herein) for human serum albumin and that comprises thesequence motif RXWD (in which X is preferably chosen from W, Y, F, S orD) and the sequence motif DVFGGG (SEQ ID NO: 129) or DAFGGG (SEQ ID NO:192).

In another aspect, the invention relates to a peptide that is specificfor (as defined herein) for human serum albumin and that comprises thesequence motif RXWD (in which X is preferably chosen from W, Y, F, S orD) and the sequence motif DVFGGGT (SEQ ID NO:133), DVFGGGS (SEQ ID NO:193) of DAFGGGT (SEQ ID NO:194).

In preferred aspects, all the above peptides are preferably further suchthat they bind better to human serum albumin than the amino acidsequence of SEQ ID NO:1 and more preferably such that they bind equallygood and more preferably better (as defined herein) to HSA than theamino acid sequence 56E4 (SEQ ID NO: 14).

In another aspect, the above peptides may be affinity matured variantsof the amino acid sequence 56E4 (SEQ ID NO:14).

Also, where the above peptides are said to contain the sequence motifRXWD, either (i) the Arg (R) residue in this motif is capable of forminga hydrogen bond with the amino acid residues Asn (N) 133 & Asn (N) 135of human serum albumin and/or capable of forming electrostaticinteractions with the main-chain oxygen atoms of the Pro (P) 134 and Leu(L) 136 residues of human serum albumin; and/or (ii) the Trp (W) residuein this motif is capable of forming electrostatic interactions with theArg (R) 138 residue of human serum albumin; and preferably both (i) and(ii) apply.

As mentioned, all these peptides may be as further described herein forthe amino acid sequences of the invention.

In another aspect, the invention relates to a peptide that is specificfor (as defined herein) for human serum albumin that comprises thesequence motif RXWD (in which X may be any amino acid, but is mostpreferably chosen from W, Y, F, S or D), in which (i) the Arg (R)residue in this motif is capable of forming a hydrogen bond with theamino acid residues Asn (N) 133 & Asn (N) 135 of human serum albuminand/or capable of forming electrostatic interactions with the main-chainoxygen atoms of the Pro (P) 134 and Leu (L) 136 residues of human serumalbumin; and/or (ii) the Trp (W) residue in this motif is capable offorming electrostatic interactions with the Arg (R) 138 residue of humanserum albumin; and preferably both (i) and (ii) apply. This peptidepreferably further contains the sequence motif FGGG, more preferably thesequence motif DVFGGG (SEQ ID NO:129), and even more preferably thesequence motif DVGGGGT (SEQ ID NO:133).

Again, such peptides are preferably further such that they bind betterto human serum albumin than the amino acid sequence of SEQ ID NO:1 andmore preferably such that they bind equally good and more preferablybetter (as defined herein) to HSA than the amino acid sequence 56E4 (SEQID NO: 14); and/or may be affinity matured variants of the amino acidsequence 56E4 (SEQ ID NO:14); and may further generally be as furtherdescribed herein.

In another aspect, the invention relates to a peptide that competes withthe peptide of SEQ ID NO:1 for binding to human serum albumin, and/orthat cross-blocks (as defined herein) the binding of the peptide of SEQID NO:1 to human serum albumin; and that binds better (as definedherein) to human serum albumin than the peptide of SEQ ID NO:1.

In another aspect, the invention relates to a peptide that competes withthe peptide of SEQ ID NO:1 for binding to human serum albumin, and/orthat cross-blocks (as defined herein) the binding of the peptide of SEQID NO:1 to human serum albumin; and that binds better (as definedherein) to human serum albumin than the peptide of SEQ ID NO:14. Such apeptide may be as further described herein.

In another aspect, the invention relates to a peptide that competes withthe peptide of SEQ ID NO:14 for binding to human serum albumin, and/orthat cross-blocks (as defined herein) the binding of the peptide of SEQID NO:14 to human serum albumin; and that binds better (as definedherein) to human serum albumin than the peptide of SEQ ID NO:1. Such apeptide may be as further described herein.

In another aspect, the invention relates to a peptide that competes withthe peptide of SEQ ID NO:14 for binding to human serum albumin, and/orthat cross-blocks (as defined herein) the binding of the peptide of SEQID NO:14 to human serum albumin; and that binds better (as definedherein) to human serum albumin than the peptide of SEQ ID NO:14. Such apeptide may be as further described herein.

The above peptides may be as further described herein; and may forexample be affinity matured variants of the peptide of SEQ ID NO:1, andmay in particular be affinity matured variants of the peptide of SEQ IDNO: 14. Also, and in particular, the above peptides may compete with thepeptide of SEQ ID NO:1 or SEQ ID NO:14, respectively, for binding to oneor more of the following amino acid residues of human serum albumin(numbering as indicated in Example 8): Asn (N) 133; Pro (P) 134; Asn (N)135; Leu (L) 136; Leu (L) 139; Arg (R) 141; Tyr (Y) 162; Glu (E) 165; He(I) 166; His (H) 170; Phe (F) 173; Phe (F) 181; Gly (G) 213; Lys (K)214; Ser (S) 217; Gln (Q) 483; and/or Lys (K) 543; more in particular toan epitope on human serum albumin that comprises either (i) the stretchof amino acid residues that comprises the residues Asn (N) 133; Pro (P)134; Asn (N) 135; Leu (L) 136; Leu (L) 139 and Arg (R) 141; and/or (ii)the stretch of amino acid residues that comprises the residues Tyr (Y)162; Glu (E) 165; Ile (I) 166; His (H) 170; Phe (F) 173; Phe (F) 181;and/or (iii) the stretch of amino acid residues that comprises theresidues Gly (G) 213; Lys (K) 214 and Ser (S) 217; and even more inparticular with a hydrophobic subpocket on human serum albumin that iscomprises (amongst others) residues the residues Leu (L) 139, Glu (E)165, Ile (I) 166, His (H) 170, Phe (F) 173, Phe (F) 181, Gly (G) 213,Lys (K) 214, Ser (S) 217 and Gln (Q) 483.

In one specific aspect, the invention relates to compounds of theinvention that comprise at least one amino acid sequence of theinvention (which may be as further described herein), and at least onesingle domain antibody (and in particular a Nanobody) against vWF, suchas one of the Nanobodies described in WO 04/062551 or WO 06/1222825).

In particular, such a compound of the invention may comprise two singledomain antibodies (and in particular two Nanobodies) against vWF (suchas two of the Nanobodies described in WO 04/062551 or WO 06/1222825),and at least one amino acid sequence of the invention. Such a compoundmay have one of the configurations exemplified above. For example, insuch a compound, the two single domain against vWF may be directlylinked to each other, or may be linked to each other via a linker thatcomprises at least one, and preferably two, amino acid sequences of theinvention.

Preferably, however, such a compound comprises two single domainantibodies (and in particular Nanobodies) against vWF that are linked toeach other via a suitable linker (that does not contain an amino acidsequence of the invention) so as to form a bivalent anti-vWF construct(for which again reference is made to WO 04/062551 or WO 06/1222825), inwhich one or more amino acid sequences of the invention (which may be inthe form of a tandem repeat as described herein) are linked to eitherthe C-terminus, to the N-terminus or to both the C-terminus and theN-terminus of the bivalent anti-vWF construct (again, optionally via asuitable linker).

More preferably, such a compound comprises two single domain antibodies(and in particular Nanobodies) against vWF (that may be different butare preferably the same) that are linked to each other via a suitablelinker (that does not contain an amino acid sequence of the invention)so as to form a bivalent anti-vWF construct, which is linked (at theC-terminus, the N-terminus or both the C-terminus and the N-terminusoptionally via a suitable linker) to a tandem repeat of amino acidsequences of the invention as described herein (in particular,comprising two amino acid sequences of the invention, linked via asuitable linker). Most preferably, such a tandem repeat is linked to theC-terminus of the bivalent anti-vWF construct.

The (preferably two) single domain antibodies (and in particularNanobodies) against vWF present in these compounds are preferablydirected against the activated confirmation of the A1 domain of vWF (seeagain WO 04/062551 and in particular WO 06/1222825). In particular, the(preferably two) single domain antibodies (and in particular Nanobodies)against vWF present in these compounds may be one of the Nanobodiesdescribed in WO 06/1222825; and more in particular humanized versions ofthe Nanobody 12A2 (SEQ ID NO: 71 of WO 06/1222825), such as thehumanized versions of 12A2 described in WO 06/1222825 (see for exampleSEQ ID NO's: 90 to 94 of WO 06/1222825, with the humanized variant of12A2H1/SEQ ID NO:90 being particularly preferred).

Some preferred, but non-limiting examples of such anti-vWF compounds ofthe invention are described and used in Examples 12-15 below. Otherpreferred Examples are as described in Example 12, but comprise ahumanized variant of 12A2 instead of 12A2 (as present in the constructsof Example 12), and in particular 12A2H1 (SEQ ID NO:90 of WO06/1222825). Another preferred example of such a compound would comprisethe anti-vWF construct of SEQ ID NO:90 of WO 06/1222825, linked at itsN-terminus (less preferred) or its C-terminus (preferred) to an aminoacid sequence of the invention, and preferably to a tandem repeat ofamino acid sequences of the invention as described herein.

A most preferred example is a compound that comprises the anti-vWFconstruct of SEQ ID NO:90 of WO 06/1222825, linked at its N-terminus(less preferred) or its C-terminus (preferred) to an amino acid sequenceof the invention, and preferably to a tandem repeat (as describedherein) that comprises two of the amino acid sequence 59C2, 59F2 and/or59H2 of the invention.

The invention also relates to a nucleotide sequence or nucleic acid thatencodes an amino acid sequence of the invention or a polypeptide of theinvention (also referred to herein as a “nucleotide sequence of theinvention” or a “nucleic acid of the invention”).

The invention also relates to a host or host cell that contains anucleotide sequence or nucleic acid of the invention and/or thatexpresses (or is capable of expressing) an amino acid sequence of theinvention or a polypeptide of the invention.

The invention also relates to methods for preparing the amino acidsequences and compounds of the invention, which methods are as furtherdescribed herein.

The invention further relates to a composition that comprises at leastone amino acid sequence of the invention or compound of the invention;and optionally one or more further suitable components or constituents.In particular, the invention relates to a pharmaceutical compositionthat comprises at least one amino acid sequence of the invention,compound of the invention, or nucleic acid of the invention; andoptionally at least one pharmaceutically acceptable carrier, diluent orexcipient.

The invention also encompasses some other methods for preparing theconstructs and compounds of the invention, which generally comprise thestep of linking at least one amino acid sequence of the invention to atleast one therapeutic moiety, optionally via one or more suitablelinkers or spacers. This may be performed in any suitable manner knownper se, for example depending on the linker(s) used (if any), and mayfor example comprise techniques for chemical linking known per se in theart, for example by formation of one or more covalent bonds. The one ormore amino acid sequences of the invention and the one or moretherapeutic moieties may be as further described herein. Again, the oneor more amino acid sequences of the invention preferably comprise adisulphide bridge as described herein.

The invention also relates to compound or construct that is obtained viaany of the above methods; and also to a pharmaceutical composition thatcomprises at least one such compound or construct and optionally atleast one pharmaceutically acceptable carrier, diluent or excipient.

The invention also relates to uses of the amino acid sequences of theinvention. Generally, these uses comprise any use known per se forbinding units, binding domains or amino acid sequences that can bind toserum proteins in general, and serum albumin in particular. Such useswill be clear to the skilled person, and not only include increasing thehalf-life to therapeutic moieties, entities or drugs; but also (or inaddition) directing therapeutic moieties, entities or drugs to parts ofthe body or tissues where serum albumin is present and/or accumulates inthe body, such as inflammation sites or joints.

The invention further relates to therapeutic uses of polypeptide orprotein constructs or fusion proteins and to pharmaceutical compositionscomprising such polypeptide or protein constructs or fusion proteins.

DETAILED DESCRIPTION OF THE INVENTION

In the present description, examples and claims:

-   a) Unless indicated otherwise herein (for example, in Example 8),    amino acid residues and positions in the amino acid sequences of the    invention will be numbered with reference to the corresponding amino    acid residues and positions in the AASYSDYDVFGGGTDFGP (SEQ ID NO:1).-   b) Unless indicated otherwise herein (for example, in Example 8),    amino acid substitutions will be mentioned with reference to the    amino acid residue present at the corresponding position in the    amino acid sequence AASYSDYDVFGGGTDFGP (SEQ ID NO:1). For example,    S3R refers to a substitution, compared to the amino acid sequence    AASYSDYDVFGGGTDFGP (SEQ ID NO:1), of the serine residue S at    position 3 into arginine (R).-   c) Unless indicated or defined otherwise, all terms used have their    usual meaning in the art, which will be clear to the skilled person.    Reference is for example made to the standard handbooks mentioned in    paragraph a) on page 46 of WO 08/020,079 of Ablynx N.V. entitled    “Amino acid sequences directed against IL-6R and polypeptides    comprising the same for the treatment of diseases and disorders    associated with Il-6 mediated signalling”.-   d) Unless indicated otherwise, the terms “immunoglobulin sequence”,    “sequence”, “nucleotide sequence” and “nucleic acid” are as    described in paragraph b) on page 46 of WO 08/020,079.-   e) Unless indicated otherwise, all methods, steps, techniques and    manipulations that are not specifically described in detail can be    performed and have been performed in a manner known per se, as will    be clear to the skilled person. Reference is for example again made    to the standard handbooks and the general background art mentioned    herein and to the further references cited therein; as well as to    for example the following reviews Presta, Adv. Drug Deliv. Rev.    2006, 58 (5-6): 640-56; Levin and Weiss, Mol. Biosyst. 2006, 2(1):    49-57; Irving et al., J. Immunol. Methods, 2001, 248(1-2), 31-45;    Schmitz et al., Placenta, 2000, 21 Suppl. A, S106-12, Gonzales et    al., Tumour Biol., 2005, 26(1), 31-43, which describe techniques for    protein engineering, such as affinity maturation and other    techniques for improving the specificity and other desired    properties of proteins such as immunoglobulins.-   f) Amino acid residues will be indicated according to the standard    three-letter or one-letter amino acid code, as mentioned in Table A;

TABLE A one-letter and three-letter amino acid code Nonpolar, AlanineAla A uncharged Valine Val V (at pH 6.0-7.0)⁽³⁾ Leucine Leu L IsoleucineIle I Phenylalanine Phe F Methionine⁽¹⁾ Met M Tryptophan Trp W ProlinePro P Polar, Glycine⁽²⁾ Gly G uncharged Serine Ser S (at pH 6.0-7.0)Threonine Thr T Cysteine Cys C Asparagine Asn N Glutamine Gln Q TyrosineTyr Y Polar, Lysine Lys K charged Arginine Arg R (at pH 6.0-7.0)Histidine⁽⁴⁾ His H Aspartate Asp D Glutamate Glu E Notes: ⁽¹⁾Sometimesalso considered to be a polar uncharged amino acid. ⁽²⁾Sometimes alsoconsidered to be a nonpolar uncharged amino acid. ⁽³⁾As will be clear tothe skilled person, the fact that an amino acid residue is referred toin this Table as being either charged or uncharged at pH 6.0 to 7.0 doesnot reflect in any way on the charge said amino acid residue may have ata pH lower than 6.0 and/or at a pH higher than 7.0; the amino acidresidues mentioned in the Table can be either charged and/or unchargedat such a higher or lower pH, as will be clear to the skilled person.⁽⁴⁾As is known in the art, the charge of a His residue is greatlydependant upon even small shifts in pH, but a His residue can generallybe considered essentially uncharged at a pH of about 6.5.

-   g) For the purposes of comparing two or more nucleotide sequences,    the percentage of “sequence identity” between a first nucleotide    sequence and a second nucleotide sequence may be calculated or    determined as described in paragraph c) on page 49 of WO 08/020,079    (incorporated herein by reference), such as by dividing [the number    of nucleotides in the first nucleotide sequence that are identical    to the nucleotides at the corresponding positions in the second    nucleotide sequence] by [the total number of nucleotides in the    first nucleotide sequence] and multiplying by [100%], in which each    deletion, insertion, substitution or addition of a nucleotide in the    second nucleotide sequence—compared to the first nucleotide    sequence—is considered as a difference at a single nucleotide    (position); or using a suitable computer algorithm or technique,    again as described in paragraph c) on pages 49 of WO 08/020,079    (incorporated herein by reference).-   h) For the purposes of comparing two or more amino acid sequences,    the percentage of “sequence identity” between a first amino acid    sequence and a second amino acid sequence (also referred to herein    as “amino acid identity”) may be calculated by dividing [the number    of amino acid residues in the first amino acid sequence that are    identical to the amino acid residues at the corresponding positions    in the second amino acid sequence] by [the total number of amino    acid residues in the first amino acid sequence] and multiplying by    [100%], in which each deletion, insertion, substitution or addition    of an amino acid residue in the second amino acid sequence—compared    to the first amino acid sequence—is considered as a difference at a    single amino acid residue (position), i.e. as an “amino acid    difference” as defined herein.    -   Alternatively, the degree of sequence identity between two amino        acid sequences may be calculated using a known computer        algorithm, such as those mentioned above for determining the        degree of sequence identity for nucleotide sequences, again        using standard settings.    -   Usually, for the purpose of determining the percentage of        “sequence identity” between two amino acid sequences in        accordance with the calculation method outlined hereinabove, the        amino acid sequence with the greatest number of amino acid        residues will be taken as the “first” amino acid sequence, and        the other amino acid sequence will be taken as the “second”        amino acid sequence.    -   Also, in determining the degree of sequence identity between two        amino acid sequences, the skilled person may take into account        so-called “conservative” amino acid substitutions, which can        generally be described as amino acid substitutions in which an        amino acid residue is replaced with another amino acid residue        of similar chemical structure and which has little or        essentially no influence on the function, activity or other        biological properties of the polypeptide. Such conservative        amino acid substitutions are well known in the art, for example        from WO 04/037999, GB-A-3 357 768, WO 98/49185, WO 00/46383 and        WO 01/09300; and (preferred) types and/or combinations of such        substitutions may be selected on the basis of the pertinent        teachings from WO 04/037999 as well as WO 98/49185 and from the        further references cited therein.    -   Such conservative substitutions preferably are substitutions in        which one amino acid within the following groups (a)-(e) is        substituted by another amino acid residue within the same        group: (a) small aliphatic, nonpolar or slightly polar residues:        Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged        residues and their (uncharged) amides: Asp, Asn, Glu and        Gln; (c) polar, positively charged residues: His, Arg and        Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val        and Cys; and (e) aromatic residues: Phe, Tyr and Trp.    -   Particularly preferred conservative substitutions are as        follows: Ala into Gly or into Ser; Arg into Lys; Asn into Gln or        into His; Asp into Glu; Cys into Ser; Gln into Asn; Glu into        Asp; Gly into Ala or into Pro; His into Asn or into Gln; He into        Leu or into Val; Leu into Ile or into Val; Lys into Arg, into        Gln or into Glu; Met into Leu, into Tyr or into Ile; Phe into        Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into        Tyr; Tyr into Trp; and/or Phe into Val, into Ile or into Leu.    -   Any amino acid substitutions applied to the polypeptides        described herein may also be based on the analysis of the        frequencies of amino acid variations between homologous proteins        of different species developed by Schulz et al., Principles of        Protein Structure, Springer-Verlag, 1978, on the analyses of        structure forming potentials developed by Chou and Fasman,        Biochemistry 13: 211, 1974 and Adv. Enzymol., 47: 45-149, 1978,        and on the analysis of hydrophobicity patterns in proteins        developed by Eisenberg et al., Proc. Natl. Acad. Sci. USA 81:        140-144, 1984; Kyte & Doolittle; J Molec. Biol. 157: 105-132,        1981, and Goldman et al., Ann. Rev. Biophys. Chem. 15: 321-353,        1986, all incorporated herein in their entirety by reference.        Information on the primary, secondary and tertiary structure of        Nanobodies® is given in the description herein and in the        general background art cited above. Also, for this purpose, the        crystal structure of a V_(HH) domain from a llama is for example        given by Desmyter et al., Nature Structural Biology, Vol. 3, 9,        803 (1996); Spinelli et al., Natural Structural Biology (1996);        3, 752-757; and Decanniere et al., Structure, Vol. 7, 4, 361        (1999). Further information about some of the amino acid        residues that in conventional V_(H) domains form the V_(H)/V_(L)        interface and potential camelizing substitutions on these        positions can be found in the prior art cited above.-   i) Amino acid sequences and nucleic acid sequences are said to be    “exactly the same” if they have 100% sequence identity (as defined    herein) over their entire length;-   j) When comparing two amino acid sequences, the term “amino acid    difference” refers to an insertion, deletion or substitution of a    single amino acid residue on a position of the first sequence,    compared to the second sequence; it being understood that two amino    acid sequences can contain one, two or more such amino acid    differences;-   k) When a nucleotide sequence or amino acid sequence is said to    “comprise” another nucleotide sequence or amino acid sequence,    respectively, or to “essentially consist of” another nucleotide    sequence or amino acid sequence, this has the meaning given in    paragraph i) on pages 51-52 of WO 08/020,079.-   l) The term “in essentially isolated form” has the meaning given to    it in paragraph j) on pages 52 and 53 of WO 08/020,079.-   m) The terms “domain” and “binding domain” have the meanings given    to it in paragraph k) on page 53 of WO 08/020,079.-   n) The terms “antigenic determinant” and “epitope”, which may also    be used interchangeably herein. have the meanings given to it in    paragraph 1) on page 53 of WO 08/020,079.-   o) As further described in paragraph m) on page 53 of WO 08/020,079,    an amino acid sequence (such as a Nanobody®, an antibody, a    polypeptide of the invention, or generally an antigen binding    protein or polypeptide or a fragment thereof) that can    (specifically) bind to, that has affinity for and/or that has    specificity for a specific antigenic determinant, epitope, antigen    or protein (or for at least one part, fragment or epitope thereof)    is said to be “against” or “directed against” or “specific for” said    antigenic determinant, epitope, antigen or protein.-   p) The terms “specificity” and “specific for” have the meaning given    to it in paragraph n) on pages 53-56 of WO 08/020,079; and as    mentioned therein refers to the number of different types of    antigens or antigenic determinants to which a particular    antigen-binding molecule or antigen-binding protein (such as a    Nanobody® or a polypeptide of the invention) molecule can bind. The    specificity of an antigen-binding protein can be determined based on    affinity and/or avidity, as described on pages 53-56 of WO    08/020,079 (incorporated herein by reference), which also describes    some preferred techniques for measuring binding between an    antigen-binding molecule (such as a Nanobody® or polypeptide of the    invention) and the pertinent antigen. Typically, antigen-binding    proteins (such as the amino acid sequences and/or compounds of the    invention) will bind to their antigen with a dissociation constant    (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or less, and preferably 10⁻⁷ to    10⁻¹² moles/liter or less and more preferably 10⁻⁸ to 10⁻¹²    moles/liter (i.e. with an association constant (K_(A)) of 10⁵ to    10¹² liter/moles or more, and preferably 10⁷ to 10¹² liter/moles or    more and more preferably 10⁸ to 10¹² liter/moles). Any K_(D) value    greater than 10⁴ mol/liter (or any K_(A) value lower than 10⁴ M⁻¹)    liters/mol is generally considered to indicate non-specific binding.    Preferably, an amino acid sequence or compound of the invention will    bind to the desired serum protein with an affinity less than 1000    nM, preferably less than 500 nM, preferably less than 200 nM, more    preferably less than 10 nM, such as less than 500 pM. Specific    binding of an antigen-binding protein to an antigen or antigenic    determinant can be determined in any suitable manner known per se,    including, for example, Scatchard analysis and/or competitive    binding assays, such as radioimmunoassays (RIA), enzyme immunoassays    (EIA) and sandwich competition assays, and the different variants    thereof known per se in the art; as well as the other techniques    mentioned herein. As will be clear to the skilled person, and as    described on pages 53-56 of WO 08/020,079, the dissociation constant    may be the actual or apparent dissociation constant. Methods for    determining the dissociation constant will be clear to the skilled    person, and for example include the techniques mentioned on pages    53-56 of WO 08/020,079-   q) The half-life of an amino acid sequence, compound or polypeptide    of the invention can generally be defined as the time taken for the    serum concentration of the amino acid sequence, compound or    polypeptide to be reduced by 50%, in vivo, for example due to    degradation of the sequence or compound and/or clearance or    sequestration of the sequence or compound by natural mechanisms. The    in vivo half-life of an amino acid sequence, compound or polypeptide    of the invention can be determined in any manner known per se, such    as by pharmacokinetic analysis. Suitable techniques will be clear to    the person skilled in the art, and may for example generally involve    the steps of suitably administering to a warm-blooded animal (i.e.    to a human or to another suitable mammal, such as a mouse, rabbit,    rat, pig, dog or a primate, for example monkeys from the genus    Macaca (such as, and in particular, cynomolgus monkeys (Macaca    fascicularis) and/or rhesus monkeys (Macaca mulatta)) and baboon    (Papio ursinus)) a suitable dose of the amino acid sequence,    compound or polypeptide of the invention; collecting blood samples    or other samples from said animal; determining the level or    concentration of the amino acid sequence, compound or polypeptide of    the invention in said blood sample; and calculating, from (a plot    of) the data thus obtained, the time until the level or    concentration of the amino acid sequence, compound or polypeptide of    the invention has been reduced by 50% compared to the initial level    upon dosing. Reference is for example made to the Experimental Part    below, as well as Dennis et al., J. Biol. Chem. 277:35035-42 (2002)    to the standard handbooks, such as Kenneth, A et al: Chemical    Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters    et al, Pharmacokinete analysis: A Practical Approach (1996).    Reference is also made to “Pharmacokinetics”, M Gibaldi & D Perron,    published by Marcel Dekker, 2nd Rev. edition (1982).    -   As will also be clear to the skilled person (see for example        pages 6 and 7 of WO 04/003019 and in the further references        cited therein), the half-life can be expressed using parameters        such as the t½-alpha, t½-beta and the area under the curve        (AUC). In the present specification, an “increase in half-life”        refers to an increase in any one of these parameters, such as        any two of these parameters, or essentially all three these        parameters. As used herein “increase in half-life” or “increased        half-life” in particular refers to an increase in the t½-beta,        either with or without an increase in the t½-alpha and/or the        AUC or both.-   r) In the context of the present invention, “modulating” or “to    modulate” generally means either reducing or inhibiting the activity    of, or alternatively increasing the activity of, a target or    antigen, as measured using a suitable in vitro, cellular or in vivo    assay. In particular, “modulating” or “to modulate” may mean either    reducing or inhibiting the activity of, or alternatively increasing    a (relevant or intended) biological activity of, a target or    antigen, as measured using a suitable in vitro, cellular or in vivo    assay (which will usually depend on the target or antigen involved),    by at least 1%, preferably at least 5%, such as at least 10% or at    least 25%, for example by at least 50%, at least 60%, at least 70%,    at least 80%, or 90% or more, compared to activity of the target or    antigen in the same assay under the same conditions but without the    presence of the construct of the invention.    -   As will be clear to the skilled person, “modulating” may also        involve effecting a change (which may either be an increase or a        decrease) in affinity, avidity, specificity and/or selectivity        of a target or antigen for one or more of its ligands, binding        partners, partners for association into a homomultimeric or        heteromultimeric form, or substrates; and/or effecting a change        (which may either be an increase or a decrease) in the        sensitivity of the target or antigen for one or more conditions        in the medium or surroundings in which the target or antigen is        present (such as pH, ion strength, the presence of co-factors,        etc.), compared to the same conditions but without the presence        of the construct of the invention. As will be clear to the        skilled person, this may again be determined in any suitable        manner and/or using any suitable assay known per se, depending        on the target or antigen involved.    -   “Modulating” may also mean effecting a change (i.e. an activity        as an agonist, as an antagonist or as a reverse agonist,        respectively, depending on the target or antigen and the desired        biological or physiological effect) with respect to one or more        biological or physiological mechanisms, effects, responses,        functions, pathways or activities in which the target or antigen        (or in which its substrate(s), ligand(s) or pathway(s) are        involved, such as its signalling pathway or metabolic pathway        and their associated biological or physiological effects) is        involved. Again, as will be clear to the skilled person, such an        action as an agonist or an antagonist may be determined in any        suitable manner and/or using any suitable (in vitro and usually        cellular or in assay) assay known per se, depending on the        target or antigen involved. In particular, an action as an        agonist or antagonist may be such that an intended biological or        physiological activity is increased or decreased, respectively,        by at least 1%, preferably at least 5%, such as at least 10% or        at least 25%, for example by at least 50%, at least 60%, at        least 70%, at least 80%, or 90% or more, compared to the        biological or physiological activity in the same assay under the        same conditions but without the presence of the construct of the        invention.    -   Modulating may for example also involve allosteric modulation of        the target or antigen; and/or reducing or inhibiting the binding        of the target or antigen to one of its substrates or ligands        and/or competing with a natural ligand, substrate for binding to        the target or antigen. Modulating may also involve activating        the target or antigen or the mechanism or pathway in which it is        involved. Modulating may for example also involve effecting a        change in respect of the folding or confirmation of the target        or antigen, or in respect of the ability of the target or        antigen to fold, to change its confirmation (for example, upon        binding of a ligand), to associate with other (sub)units, or to        disassociate. Modulating may for example also involve effecting        a change in the ability of the target or antigen to transport        other compounds or to serve as a channel for other compounds        (such as ions).    -   Modulating may be reversible or irreversible, but for        pharmaceutical and pharmacological purposes will usually be in a        reversible manner.-   s) In respect of a target or antigen, the term “interaction site” on    the target or antigen means a site, epitope, antigenic determinant,    part, domain or stretch of amino acid residues on the target or    antigen that is a site for binding to a ligand, receptor or other    binding partner, a catalytic site, a cleavage site, a site for    allosteric interaction, a site involved in multimerization (such as    homomerization or heterodimerization) of the target or antigen; or    any other site, epitope, antigenic determinant, part, domain or    stretch of amino acid residues on the target or antigen that is    involved in a biological action or mechanism of the target or    antigen. More generally, an “interaction site” can be any site,    epitope, antigenic determinant, part, domain or stretch of amino    acid residues on the target or antigen to which an amino acid    sequence or polypeptide of the invention can bind such that the    target or antigen (and/or any pathway, interaction, signalling,    biological mechanism or biological effect in which the target or    antigen is involved) is modulated (as defined herein).-   t) An amino acid sequence or polypeptide is said to be “specific    for” a first target or antigen compared to a second target or    antigen when is binds to the first antigen with an affinity (as    described above, and suitably expressed as a K_(D) value, K_(A)    value, K_(off) rate and/or K_(on) rate) that is at least 10 times,    such as at least 100 times, and preferably at least 1000 times, and    up to 10,000 times or more better than the affinity with which said    amino acid sequence or polypeptide binds to the second target or    polypeptide. For example, the first antigen may bind to the target    or antigen with a K_(D) value that is at least 10 times less, such    as at least 100 times less, and preferably at least 1000 times less,    such as 10,000 times less or even less than that, than the K_(D)    with which said amino acid sequence or polypeptide binds to the    second target or polypeptide. Preferably, when an amino acid    sequence or polypeptide is “specific for” a first target or antigen    compared to a second target or antigen, it is directed against (as    defined herein) said first target or antigen, but not directed    against said second target or antigen.-   u) An amino acid sequence is said to be “cross-reactive” for two    different antigens or antigenic determinants (such as serum albumin    from two different species of mammal, such as human serum albumin    and cyno serum albumin) if it is specific for (as defined herein)    both these different antigens or antigenic determinants.-   v) By binding that is “essentially independent of the pH” is    generally meant herein that the association constant (K_(A)) of the    amino acid sequence with respect to the serum protein (such as serum    albumin) at the pH value(s) that occur in a cell of an animal or    human body (as further described herein) is at least 5%, such as at    least 10%, preferably at least 25%, more preferably at least 50%,    even more preferably at least 60%, such as even more preferably at    least 70%, such as at least 80% or 90% or more (or even more than    100%, such as more than 110%, more than 120% or even 130% or more,    or even more than 150%, or even more than 200%) of the association    constant (K_(A)) of the amino acid sequence with respect to the same    serum protein at the pH value(s) that occur outside said cell.    Alternatively, by binding that is “essentially independent of the    pH” is generally meant herein that the k_(off) rate (measured by    Biacore—see e.g. Experiment 2) of the amino acid sequence with    respect to the serum protein (such as serum albumin) at the pH    value(s) that occur in a cell of an animal or human body (as e.g.    further described herein, e.g. pH around 5.5, e.g. 5.3 to 5.7) is at    least 5%, such as at least 10%, preferably at least 25%, more    preferably at least 50%, even more preferably at least 60%, such as    even more preferably at least 70%, such as at least 80% or 90% or    more (or even more than 100%, such as more than 110%, more than 120%    or even 130% or more, or even more than 150%, or even more than    200%) of the k_(off) rate of the amino acid sequence with respect to    the same serum protein at the pH value(s) that occur outside said    cell, e.g. pH 7.2 to 7.4. By “the pH value(s) that occur in a cell    of an animal or human body” is meant the pH value(s) that may occur    inside a cell, and in particular inside a cell that is involved in    the recycling of the serum protein. In particular, by “the pH    value(s) that occur in a cell of an animal or human body” is meant    the pH value(s) that may occur inside a (sub)cellular compartment or    vesicle that is involved in recycling of the serum protein (e.g. as    a result of pinocytosis, endocytosis, transcytosis, exocytosis and    phagocytosis or a similar mechanism of uptake or internalization    into said cell), such as an endosome, lysosome or pinosome.-   w) The terms “cross-block”, “cross-blocked” and “cross-blocking” are    used interchangeably herein to mean the ability of an amino acid    sequence or other binding agents (such as a Nanobody, polypeptide or    compound or construct of the invention) to interfere with the    binding of other amino acid sequences or binding agents of the    invention to a given target. The extend to which an amino acid    sequence or other binding agents of the invention is able to    interfere with the binding of another to the relevant, and therefore    whether it can be said to cross-block according to the invention,    can be determined using competition binding assays. One particularly    suitable quantitative cross-blocking assay uses a Biacore machine    which can measure the extent of interactions using surface plasmon    resonance technology. Another suitable quantitative cross-blocking    assay uses an ELISA-based approach to measure competition between    amino acid sequences or other binding agents in terms of their    binding to the target.    -   The following generally describes a suitable Biacore assay for        determining whether an amino acid sequence or other binding        agent cross-blocks or is capable of cross-blocking according to        the invention. It will be appreciated that the assay can be used        with any of the amino acid sequences or other binding agents        described herein. The Biacore machine (for example the        Biacore 3000) is operated in line with the manufacturer's        recommendations. Thus in one cross-blocking assay, the target        protein is coupled to a CM5 Biacore chip using standard amine        coupling chemistry to generate a surface that is coated with the        target. Typically 200-800 resonance units of the target would be        coupled to the chip (an amount that gives easily measurable        levels of binding but that is readily saturable by the        concentrations of test reagent being used). Two test amino acid        sequences (termed A* and B*) to be assessed for their ability to        cross-block each other are mixed at a one to one molar ratio of        binding sites in a suitable buffer to create the test mixture.        When calculating the concentrations on a binding site basis the        molecular weight of an amino acid sequence is assumed to be the        total molecular weight of the amino acid sequence divided by the        number of target binding sites on that amino acid sequence. The        concentration of each amino acid sequence in the test mix should        be high enough to readily saturate the binding sites for that        amino acid sequence on the target molecules captured on the        Biacore chip. The amino acid sequences in the mixture are at the        same molar concentration (on a binding basis) and that        concentration would typically be between 1.00 and 1.5 micromolar        (on a binding site basis). Separate solutions containing A*        alone and B* alone are also prepared. A* and B* in these        solutions should be in the same buffer and at the same        concentration as in the test mix. The test mixture is passed        over the target-coated Biacore chip and the total amount of        binding recorded. The chip is then treated in such a way as to        remove the bound amino acid sequences without damaging the        chip-bound target. Typically this is done by treating the chip        with 30 mM HCl for 60 seconds. The solution of A* alone is then        passed over the target-coated surface and the amount of binding        recorded. The chip is again treated to remove all of the bound        amino acid sequences without damaging the chip-bound target. The        solution of B* alone is then passed over the target-coated        surface and the amount of binding recorded. The maximum        theoretical binding of the mixture of A* and B* is next        calculated, and is the sum of the binding of each amino acid        sequence when passed over the target surface alone. If the        actual recorded binding of the mixture is less than this        theoretical maximum then the two amino acid sequences are        cross-blocking each other. Thus, in general, a cross-blocking        amino acid sequence or other binding agent according to the        invention is one which will bind to the target in the above        Biacore cross-blocking assay such that, during the assay and in        the presence of a second amino acid sequence or other binding        agent of the invention, the recorded binding is between 80% and        0.1% (e.g. 80% to 4%) of the maximum theoretical binding,        specifically between 75% and 0.1% (e.g. 75% to 4%) of the        maximum theoretical binding, and more specifically between 70%        and 0.1% (e.g. 70% to 4%) of maximum theoretical binding (as        just defined above) of the two amino acid sequences or binding        agents in combination. The Biacore assay described above is a        primary assay used to determine if amino acid sequences or other        binding agents cross-block each other according to the        invention. On rare occasions particular amino acid sequences or        other binding agents may not bind to target coupled via amine        chemistry to a CM5 Biacore chip (this usually occurs when the        relevant binding site on target is masked or destroyed by the        coupling to the chip). In such cases cross-blocking can be        determined using a tagged version of the target, for example a        N-terminal His-tagged version. In this particular format, an        anti-His amino acid sequence would be coupled to the Biacore        chip and then the His-tagged target would be passed over the        surface of the chip and captured by the anti-His amino acid        sequence. The cross blocking analysis would be carried out        essentially as described above, except that after each chip        regeneration cycle, new His-tagged target would be loaded back        onto the anti-His amino acid sequence coated surface. In        addition to the example given using N-terminal His-tagged        target, C-terminal His-tagged target could alternatively be        used. Furthermore, various other tags and tag binding protein        combinations that are known in the art could be used for such a        cross-blocking analysis (e.g. HA tag with anti-HA antibodies;        FLAG tag with anti-FLAG antibodies; biotin tag with        streptavidin).    -   The following generally describes an ELISA assay for determining        whether an amino acid sequence or other binding agent directed        against a target cross-blocks or is capable of cross-blocking as        defined herein. It will be appreciated that the assay can be        used with any of the amino acid sequences (or other binding        agents such as polypeptides of the invention) described herein.        The general principal of the assay is to have an amino acid        sequence or binding agent that is directed against the target        coated onto the wells of an ELISA plate. An excess amount of a        second, potentially cross-blocking, anti-target amino acid        sequence is added in solution (i.e. not bound to the ELISA        plate). A limited amount of the target is then added to the        wells. The coated amino acid sequence and the amino acid        sequence in solution compete for binding of the limited number        of target molecules. The plate is washed to remove excess target        that has not been bound by the coated amino acid sequence and to        also remove the second, solution phase amino acid sequence as        well as any complexes formed between the second, solution phase        amino acid sequence and target. The amount of bound target is        then measured using a reagent that is appropriate to detect the        target. An amino acid sequence in solution that is able to        cross-block the coated amino acid sequence will be able to cause        a decrease in the number of target molecules that the coated        amino acid sequence can bind relative to the number of target        molecules that the coated amino acid sequence can bind in the        absence of the second, solution phase, amino acid sequence. In        the instance where the first amino acid sequence, e.g. an Ab-X,        is chosen to be the immobilized amino acid sequence, it is        coated onto the wells of the ELISA plate, after which the plates        are blocked with a suitable blocking solution to minimize        non-specific binding of reagents that are subsequently added. An        excess amount of the second amino acid sequence, i.e. Ab-Y, is        then added to the ELISA plate such that the moles of Ab-Y target        binding sites per well are at least 10 fold higher than the        moles of Ab-X target binding sites that were used, per well,        during the coating of the ELISA plate. Target is then added such        that the moles of target added per well are at least 25-fold        lower than the moles of Ab-X target binding sites that were used        for coating each well. Following a suitable incubation period        the ELISA plate is washed and a reagent for detecting the target        is added to measure the amount of target specifically bound by        the coated anti[target amino acid sequence (in this case Ab-X).        The background signal for the assay is defined as the signal        obtained in wells with the coated amino acid sequence (in this        case Ab-X), second solution phase amino acid sequence (in this        case Ab-Y), target buffer only (i.e. without target) and target        detection reagents. The positive control signal for the assay is        defined as the signal obtained in wells with the coated amino        acid sequence (in this case Ab-X), second solution phase amino        acid sequence buffer only (i.e. without second solution phase        amino acid sequence), target and target detection reagents. The        ELISA assay may be run in such a manner so as to have the        positive control signal be at least 6 times the background        signal. To avoid any artifacts (e.g. significantly different        affinities between Ab-X and Ab-Y for the target) resulting from        the choice of which amino acid sequence to use as the coating        amino acid sequence and which to use as the second (competitor)        amino acid sequence, the cross-blocking assay may to be run in        two formats: 1) format 1 is where Ab-X is the amino acid        sequence that is coated onto the ELISA plate and Ab-Y is the        competitor amino acid sequence that is in solution and 2) format        2 is where Ab-Y is the amino acid sequence that is coated onto        the ELISA plate and Ab-X is the competitor amino acid sequence        that is in solution. Ab-X and Ab-Y are defined as cross-blocking        if, either in format 1 or in format 2, the solution phase        anti-target amino acid sequence is able to cause a reduction of        between 60% and 100%, specifically between 70% and 100%, and        more specifically between 80% and 100%, of the target detection        signal {i.e. the amount of target bound by the coated amino acid        sequence) as compared to the target detection signal obtained in        the absence of the solution phase anti-target amino acid        sequence (i.e. the positive control wells).-   x) Any Figures, Sequence Listing and the Experimental Part/Examples    are only given to further illustrate the invention and should not be    interpreted or construed as limiting the scope of the invention    and/or of the appended claims in any way, unless explicitly    indicated otherwise herein.

For a general description of heavy chain antibodies and the variabledomains thereof, reference is inter alia made to the prior art citedherein, to the review article by Muyldermans in Reviews in MolecularBiotechnology 74 (2001), 277-302; as well as to the following patentapplications, which are mentioned as general background art: WO94/04678, WO 95/04079 and WO 96/34103 of the Vrije Universiteit Brussel;WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO01/40310, WO 01/44301, EP 1134231 and WO 02/48193 of Unilever; WO97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 ofthe Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 ofAlgonomics N.V. and Ablynx N.V.; WO 01/90190 by the National ResearchCouncil of Canada; WO 03/025020 (=EP 1 433 793) by the Institute ofAntibodies; as well as WO 04/041867, WO 04/041862, WO 04/041865, WO04/041863, WO 04/062551, WO 05/044858, WO 06/40153, WO 06/079372, WO06/122786, WO 06/122787 and WO 06/122825, by Ablynx N.V. and the furtherpublished patent applications by Ablynx N.V. Reference is also made tothe further prior art mentioned in these applications, and in particularto the list of references mentioned on pages 41-43 of the Internationalapplication WO 06/040153, which list and references are incorporatedherein by reference.

The amino acid sequences of the invention may be prepared in a mannerknown per se. For example, a desired amino acid sequence may be preparedby peptide synthesis or by suitably expressing a nucleic acid encodingsaid amino acid sequence. A desired nucleotide sequence may be preparedby techniques of nucleic acid synthesis known per se.

One method for preparing the amino acid sequences or polypeptides of theinvention generally comprises at least the step of:

-   a) expressing a nucleotide sequence or nucleic acid of the    invention;    and optionally further comprises:-   b) isolating the amino acid sequence of the invention or the    polypeptide of the invention, respectively, so expressed.

Another method for preparing the amino acid sequences or polypeptides ofthe invention generally comprises at least the step of:

-   a) cultivating or maintaining a host or host cell as described    herein under conditions such that said host or host cell produces an    amino acid sequence or polypeptide of the invention;    and optionally further comprising:-   b) isolating the amino acid sequence of the invention or polypeptide    of the invention respectively, thus obtained.

Where an amino acid sequence of the invention is to be used in aconstrained format (i.e. comprising a disulphide bridge between theflanking sequences that flank the amino acid sequence of the invention),the above methods may also comprise a further step of forming such adisulphide bridge, as further described in PCT/EP2007/063348.

The invention also relates to the amino acid sequences, compounds,construct or polypeptides obtained via the above methods.

The amino acid sequences disclosed herein can be used with advantage asa fusion partner in order to increase the half-life of therapeuticmoieties such as proteins, compounds (including, without limitation,small molecules) or other therapeutic entities. Thus, in another aspect,the invention provides amino acid sequences that can be used as smallpeptides or peptide moieties for linking or fusing to a therapeuticcompound in order to increase the half-life thereof, and constructs andfusion proteins comprising such peptides or peptide moieties, that canbind to a serum protein in such a way that, when the amino acidsequence, construct, or fusion protein of the invention is bound to aserum protein molecule, the half-life of the serum protein molecule isnot (significantly) reduced (i.e. compared to the half-life of the serumprotein molecule when the amino acid sequence, construct, or fusionprotein is not bound thereto). In this aspect of the invention, by “notsignificantly reduced” is meant that the half-life of the serum proteinmolecule (as measured using a suitable technique known per se) is notreduced by more than 50%, preferably not reduced by more than 30%, evenmore preferably not reduced by more than 10%, such as not reduced bymore than 5%, or essentially not reduced at all.

In another preferred, but non-limiting aspect, the amino acid sequencesof the invention are preferably such that they bind to or otherwiseassociate with human serum albumin in such a way that, when the aminoacid sequences are bound to or otherwise associated with a human serumalbumin, the amino acid sequences exhibit a serum half-life in human ofat least about 9 days (such as about 9 to 14 days), preferably at leastabout 10 days (such as about 10 to 15 days), or at least about 11 days(such as about 11 to 16 days), more preferably at least about 12 days(such as about 12 to 18 days or more), or more than 14 days (such asabout 14 to 19 days).

In another aspect, the invention provides polypeptide or proteinconstructs that comprise or essentially consist of an amino acidsequence as disclosed herein.

The invention also relates to a compound or construct which comprises atleast one amino acid sequence of the invention and at least onetherapeutic moiety (also referred to herein as “compounds of theinvention”).

For example, and without limitation, a compound of the invention maycomprise the at least one therapeutic moiety, that is linked to one,two, three, four or more amino acid sequences of the invention. Forexample, when the therapeutic moiety is a protein or polypeptide, theone or more amino acid sequences of the invention may be linked to theC-terminus of the protein or polypeptide (either directly or via asuitable spacer or linker); to the N-terminus of the protein orpolypeptide (again either directly or via a suitable spacer or linker);or both to the C-terminus and the N-terminus. When a compound of theinvention comprises two or more amino acid sequences of the invention,these may be the same or different.

The therapeutic moiety may also be linked (either at its C-terminus, itsN-terminus, or both, and again either directly or via a suitable spaceror linker) to a multimer or concatamer that comprises at least two (suchas two, three or four) amino acid sequences of the invention (which maybe the same or different), that may either be linked directly to eachother, or via a suitable linker or spacer. Such (bivalent, trivalent ormultivalent) multimers or concatamers (and nucleotide sequences encodingthe same, as well as compounds of the invention comprising the same)form a further aspect of the invention, and may bind to serum albuminwith a higher avidity than a monomeric amino acid sequence of theinvention.

Also, when a compound of the invention comprises two or more therapeuticmoieties, each of these therapeutic moieties (or both) may be linked toone or more amino acid sequences of the invention, as further describedherein. Also, the two or more therapeutic moieties may be linked to eachother via a linker that comprises or essentially consists of one or moreamino acid sequences of the invention (and optionally further linkingamino acid sequences), and such a linker (as well as compounds of theinvention comprising the same) form a further aspect of the invention.

In one aspect, the therapeutic moiety is directed against a desiredantigen or target, is capable of binding to a desired antigen (and inparticular capable of specifically binding to a desired antigen), and/oris capable of interacting with a desired target. In another embodiment,the at least one therapeutic moiety comprises or essentially consists ofa therapeutic protein or polypeptide. In a further embodiment, the atleast one therapeutic moiety comprises or essentially consists of animmunoglobulin or immunoglobulin sequence (including but not limited toa fragment of an immunoglobulin), such as an antibody or an antibodyfragment (including but not limited to an ScFv fragment or Fabfragment). In yet another embodiment, the at least one therapeuticmoiety comprises or essentially consists of an antibody variable domain,such as a heavy chain variable domain or a light chain variable domain.

In one preferred, but non-limiting aspect, the one or more therapeuticmoieties or entities may be one or more binding units (as defined inPCT/EP2007/063348) or binding domains (as defined herein), i.e. bindingunits or domain that are capable of binding to a desired target, antigenor antigenic determinant (such as a therapeutically relevant target). Assuch, the compound of the invention may be a monovalent, bivalent,bispecific, multivalent or multispecific construct (as defined inPCT/EP2007/063348). The binding unit may generally comprise ascaffold-based binding unit or domain, such as binding scaffolds basedon or derived from immunoglobulins (i.e. other than the immunoglobulinsequences already described herein), protein scaffolds derived fromprotein A domains (such as Affibodies™) tendamistat, fibronectin,lipocalin, CTLA-4, T-cell receptors, designed ankyrin repeats, avimersand PDZ domains (Binz et al., Nat. Biotech 2005, Vol 23:1257), andbinding moieties based on DNA or RNA including but not limited to DNA orRNA aptamers (Ulrich et al., Comb Chem High Throughput Screen 20069(8):619-32).

The amino acid sequences of the invention may also be linked to one ofthe “polypeptide drugs” referred to in the International application WO05/118642 (Domantis Ltd.) or the International application 06/059106(Domantis Ltd.); such as to one of the polypeptide drugs that arementioned on pages 45 to 50 of WO 05/118642; antagonists of theinterleukin 1 receptor (see pages 11-12 of WO 05/118642) includingfunctional variants of IL-1ra; saporins (see pages 12-14 of WO05/118642); the anticancer peptides listed in Table 8 of WO 05/118642;and insulinotropic agents or analogues thereof such as GLP-1 or GLP-1analogues (see 06/059106).

In a preferred aspect, the at least one therapeutic moiety comprises oressentially consists of at least one domain antibody or single domainantibody, “dAb” or Nanobody®. Thus, for example, in a compound of theinvention, one or more amino acid sequences of the invention may befused or linked to one or more domain antibodies, single domainantibodies, “dAb's” or Nanobodies®, such that the resulting compound ofthe invention is a monovalent, bivalent, multivalent, bispecific ormultispecific construct (in which the terms “monovalent”, “bivalent”,“multivalent”, “bispecific” and “multispecific” are as described inPCT/EP2007/063348 or in the patent applications of Ablynx N.V. citedabove).

Thus, one embodiment of the invention relates to a protein orpolypeptide construct or fusion protein that comprises or essentiallyconsists of at least one amino acid sequence of the invention and atleast one immunoglobulin sequence, such as a domain antibody, a singledomain antibody, a “dAb” or a Nanobody®.

Generally, a compound of the invention preferably has a half-life thatis more than 1 hour, preferably more than 2 hours, more preferably ofmore than 6 hours, such as of more than 12 hours, and for example ofabout one day, two days, one week, two weeks or three weeks, andpreferably no more than 2 months, although the latter may be lesscritical.

Preferably, the compounds or polypeptides of the invention that compriseat least one amino acid sequence of the invention and at least onetherapeutic moiety preferably have a half-life that is at least 1.5times, preferably at least 2 times, such as at least 5 times, forexample at least 10 times or more than 20 times, greater than thehalf-life of the therapeutic moiety per se. For example, the compoundsor polypeptides of the invention may have a half-life that is increasedwith more than 1 hours, preferably more than 2 hours, more preferablymore than 6 hours, such as more than 12 hours, or even more than 24, 48or 72 hours, compared to the therapeutic moiety per se.

In a preferred, but non-limiting aspect of the invention, such compoundsor polypeptides of the invention have a serum half-life that isincreased with more than 1 hours, preferably more than 2 hours, morepreferably more than 6 hours, such as more than 12 hours, or even morethan 24, 48 or 72 hours, compared to the therapeutic moiety per se.

The invention also relates to nucleotide sequences or nucleic acids thatencode amino acid sequences, compounds, proteins, polypeptides, fusionproteins, or multivalent or multispecific constructs described herein.The invention further includes genetic constructs that include theforegoing nucleotide sequences or nucleic acids and one or more elementsfor genetic constructs known per se. The genetic construct may be in theform of a plasmid or vector. Such and other genetic constructs are knownby those skilled in the art.

The invention also relates to hosts or host cells that contain suchnucleotide sequences or nucleic acids, and/or that express (or arecapable of expressing) amino acid sequences, compounds, proteins,polypeptides, fusion proteins, or multivalent or multispecificconstructs described herein. Again, such hosts or host cells are knownby those skilled in the art.

The invention also generally relates to a method for preparing aminoacid sequences, compounds, proteins, polypeptides, fusion proteins, ormultivalent or multispecific constructs as described herein, whichmethod comprises cultivating or maintaining a host cell as describedherein under conditions such that said host cell produces or expressesan amino acid sequence, compound, protein, polypeptide, fusion protein,or multivalent or multispecific construct as described herein, andoptionally further comprises isolating the amino acid sequence,compound, protein, polypeptide, fusion protein, or multivalent ormultispecific construct so produced. Again, such methods can beperformed as generally described in the co-pending patent applicationsby Ablynx N.V. described herein, such as WO 04/041862 or WO 06/122825.

The invention also encompasses medical uses and methods of treatmentencompassing the amino acid sequence, compound, or multivalent andmultispecific compound of the invention, wherein said medical use ormethod is characterized in that said medicament is suitable foradministration at intervals of at least about 50% of the naturalhalf-life of human serum albumin.

The invention also relates to methods for extending or increasing theserum half-life of a therapeutic (i.e. a therapeutic moiety, compound,protein or other therapeutic entity). The methods include contacting thetherapeutic with any of the foregoing amino acid sequences, such thatthe therapeutic is bound to or otherwise associated with the amino acidsequences, compounds, fusion proteins or constructs of the invention. Insome embodiments, the therapeutic is a biological therapeutic,preferably a peptide or a polypeptide, in which case the step ofcontacting the therapeutic can include preparing a fusion protein bylinking the peptide or polypeptide with the amino acid sequence,compound, fusion proteins or constructs of the invention.

These methods can further include administering the therapeutic to asubject after the therapeutic is bound to or associated with the aminoacid sequence, compound, fusion protein or construct of the invention.In such methods, the serum half-life of the therapeutic is at least 1.5times the half-life of therapeutic per se, or is increased by at least 1hour (such as by at least 6 hours, preferably at least 12 hours, morepreferably at least 1 day, such as more than 2 days, or even more than 5days or more) compared to the half-life of therapeutic per se. In somepreferred embodiments, the serum half-life of the therapeutic is atleast 2 times, at least 5 times, at least 10 times, or more than 20times greater than the half-life of the corresponding therapeutic moietyper se. In other preferred embodiments, the serum half-life of thetherapeutic is increased by more than 2 hours, more than 6 hours or morethan 12 hours compared to the half-life of the corresponding therapeuticmoiety per se.

In the above methods, the serum half-life of the therapeutic ispreferably increased or extended such that said serum half-life (i.e. ofthe compound of the invention thus obtained) is longer than the serumhalf-life of a corresponding compound or construct that comprises thetherapeutic and the amino acid sequence of SEQ ID NO:1 (i.e. instead ofthe amino acid sequence of the invention). Preferably, the serumhalf-life of the compound of the invention is at least 5% longer,preferably at least 10% longer, more preferably at least 25% longer, oreven more preferably at least than 50% longer, such as more than 100%longer or even more improved, compared to the serum half-life of acorresponding compound or construct that comprises the therapeutic andthe amino acid sequence of SEQ ID NO:1 (i.e. instead of the amino acidsequence of the invention).

For example, in such methods, the serum half-life of the compound of theinvention may be at least 1.1, such as at least 1.2 times, morepreferably at least 1.5 times the half-life of the correspondingcompound or construct that comprises the therapeutic and the amino acidsequence of SEQ ID NO:1 (i.e. instead of the amino acid sequence of theinvention), and/or may be increased by at least 1 hour (such as by atleast 6 hours, preferably at least 12 hours, more preferably at least 1day, such as more than 2 days, or even more than 5 days or more)compared to the half-life of a corresponding compound or construct thatcomprises the therapeutic and the amino acid sequence of SEQ ID NO:1(i.e. instead of the amino acid sequence of the invention). In somepreferred embodiments, the serum half-life of the compound of theinvention is at least 2 times, at least 3 times or at least 5 timesgreater than the half-life of the corresponding compound or constructthat comprises the therapeutic and the amino acid sequence of SEQ IDNO:1 (i.e. instead of the amino acid sequence of the invention).

In another aspect, the invention relates to a method for modifying atherapeutic such that the desired therapeutic level of said therapeuticis, upon suitable administration of said therapeutic so as to achievesaid desired therapeutic level, maintained for a prolonged period oftime.

The methods include contacting the therapeutic with any of the foregoingamino acid sequences, such that the therapeutic is bound to or otherwiseassociated with the amino acid sequences, compounds, fusion proteins orconstructs of the invention. In some embodiments, the therapeutic is abiological therapeutic, preferably a peptide or polypeptide, in whichcase the step of contacting the therapeutic can include preparing afusion protein by linking the peptide or polypeptide with the amino acidsequence, compound, fusion protein, or constructs of the invention.

These methods can further include administering the therapeutic to asubject after the therapeutic is bound to or otherwise associated withthe amino acid sequence, compound, fusion protein, or construct of theinvention, such that the desired therapeutic level is achieve upon suchadministration. In such methods, the time that the desired therapeuticlevel of said therapeutic is maintained upon such administration is atleast 1.5 times the half-life of therapeutic per se, or is increased byat least 1 hour compared to the half-life of therapeutic per se. In somepreferred embodiments, the time that the desired therapeutic level ofsaid therapeutic is maintained upon such administration is at least 2times, at least 5 times, at least 10 times or more than 20 times greaterthan the half-life of the corresponding therapeutic moiety per se. Inother preferred embodiments, the time that the desired therapeutic levelof said therapeutic is maintained upon such administration is increasedby more than 2 hours, more than 6 hours or more than 12 hours comparedto the half-life of the corresponding therapeutic moiety per se.

Preferably, the time that the desired therapeutic level of saidtherapeutic is maintained upon such administration is increased suchthat the therapeutic can be administered at a frequency that is asdefined herein for the compounds of the invention.

In the above methods, the time that the desired therapeutic level ofsaid therapeutic is maintained is preferably increased or extended suchthat said serum half-life (i.e. of the compound of the invention thusobtained) is longer than the time that the desired therapeutic level ofsaid therapeutic is maintained by a corresponding compound or constructthat comprises the therapeutic and the amino acid sequence of SEQ IDNO:1 (i.e. instead of the amino acid sequence of the invention).Preferably, the time that the desired therapeutic level of saidtherapeutic is maintained is at least 5% longer, preferably at least 10%longer, more preferably at least 25% longer, or even more preferably atleast than 50% longer, such as more than 100% longer or even moreimproved, compared to the time that the desired therapeutic level ofsaid therapeutic is maintained by a corresponding compound or constructthat comprises the therapeutic and the amino acid sequence of SEQ IDNO:1 (i.e. instead of the amino acid sequence of the invention).

For example, in such methods, the time that the desired therapeuticlevel of said therapeutic is maintained may be at least 1.1, such as atleast 1.2 times, more preferably at least 1.5 times the time that thedesired therapeutic level of said therapeutic is maintained by acorresponding compound or construct that comprises the therapeutic andthe amino acid sequence of SEQ ID NO:1 (i.e. instead of the amino acidsequence of the invention), and/or may be increased by at least 1 hour(such as by at least 6 hours, preferably at least 12 hours, morepreferably at least 1 day, such as more than 2 days, or even more than 5days or more) compared to the time that the desired therapeutic level ofsaid therapeutic is maintained by a corresponding compound or constructthat comprises the therapeutic and the amino acid sequence of SEQ IDNO:1 (i.e. instead of the amino acid sequence of the invention). In somepreferred embodiments, the time that the desired therapeutic level ofsaid therapeutic is maintained is at least 2 times, at least 3 times orat least 5 times greater than the time that the desired therapeuticlevel of said therapeutic is maintained by a corresponding compound orconstruct that comprises the therapeutic and the amino acid sequence ofSEQ ID NO:1 (i.e. instead of the amino acid sequence of the invention).

In another aspect, the invention relates to the use of a compound of theinvention (as defined herein) for the production of a medicament thatincreases and/or extends the level of the therapeutic agent in saidcompound or construct in the serum of a patient such that saidtherapeutic agent in said compound or construct is capable of beingadministered at a lower dose as compared to the therapeutic agent alone(i.e. at essentially the same frequency of administration).

The invention also relates to a pharmaceutical composition thatcomprises at least one amino acid sequence, compound, protein,polypeptide, fusion protein, or multivalent or multispecific constructas described herein, and optionally at least one pharmaceuticallyacceptable carrier, diluent or excipient. Such preparations, carriers,excipients and diluents may generally be as described in the co-pendingpatent applications by Ablynx N.V. described herein, such as WO04/041862 or WO 06/122825.

However, since the amino acid sequences, compounds, proteins,polypeptides, fusion proteins, or multivalent or multispecificconstructs described herein have an increased half-life, they arepreferably administered to the circulation. As such, they can beadministered in any suitable manner that allows the amino acidsequences, compounds, proteins, polypeptides, fusion proteins, ormultivalent or multispecific constructs to enter the circulation, suchas intravenously, via injection or infusion, or in any other suitablemanner (including oral administration, administration through the skin,intranasal administration, administration via the lungs, etc). Suitablemethods and routes of administration will be clear to the skilledperson, again for example also from the teaching of WO 04/041862 or WO06/122825.

Thus, in another aspect, the invention relates to a method for theprevention and/or treatment of at least one disease or disorder that canbe prevented or treated by the use of amino acid sequences, compounds,proteins, polypeptides, fusion proteins, or multivalent or multispecificconstructs described herein, which method comprises administering, to asubject in need thereof, a pharmaceutically active amount of a aminoacid sequences, compounds, proteins, polypeptides, fusion proteins, ormultivalent or multispecific constructs of the invention, and/or of apharmaceutical composition comprising the same. As will be clear to theskilled person, the diseases and disorders that can be prevented ortreated by the use of amino acid sequences, compounds, proteins,polypeptides, fusion proteins, or multivalent or multispecificconstructs described herein will generally be the same as the diseasesand disorders that can be prevented or treated by the use of thetherapeutic moiety that is present in the amino acid sequences,compounds, proteins, polypeptides, fusion proteins, or multivalent ormultispecific constructs of the invention.

In the context of the present invention, the term “prevention and/ortreatment” not only comprises preventing and/or treating a disease, butalso generally comprises preventing the onset of a disease, slowing orreversing the progress of a disease, preventing or slowing the onset ofone or more symptoms associated with a disease, reducing and/oralleviating one or more symptoms associated with a disease, reducing theseverity and/or the duration of a disease and/or of any symptomsassociated therewith and/or preventing a further increase in theseverity of a disease and/or of any symptoms associated therewith,preventing, reducing or reversing any physiological damage caused by adisease, and generally any pharmacological action that is beneficial tothe patient being treated.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal, and more in particular a human being. As will beclear to the skilled person, the subject to be treated will inparticular be a person suffering from, or at risk from, the diseases anddisorders mentioned herein.

More specifically, the present invention relates to a method oftreatment wherein the frequency of administering the amino acidsequence, compound, fusion protein or construct of the invention is atleast 50% of the natural half-life of serum albumin in said mammal (i.e.in the case of man, of human serum albumin), preferably at least 60%,preferably at least 70%, more preferably at least 80%, and mostpreferably at least 90%.

Specific frequencies of administration to a mammal, which are within thescope of the present invention are at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or at least 100% of the natural half-life ofserum albumin in said mammal as defined above.

In other words, specific frequencies of administration, which are withinthe scope of the present invention are every 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, or 19 days.

Without limitation, the frequencies of administration referred to aboveare in particular suited for maintaining a desired level of the aminoacid sequence, compound, fusion protein or construct in the serum of thesubject treated with the amino acid sequence, compound, fusion protein,or construct, optionally after administration of one or more (initial)doses that are intended to establish said desired serum level. As willbe clear to the skilled person, the desired serum level may inter aliabe dependent on the amino acid sequence, compound, fusion protein, orconstruct used and/or the disease to be treated. The clinician orphysician will be able to select the desired serum level and to selectthe dose(s) and/or amount(s) to be administered to the subject to betreated in order to achieve and/or maintain the desired serum level insaid subject, when the amino acid sequence, compound, fusion protein, orconstruct of the invention is administered at the frequencies mentionedherein.

In another embodiment, the invention relates to a method forimmunotherapy, and in particular for passive immunotherapy, which methodcomprises administering, to a subject suffering from or at risk of thediseases and disorders mentioned herein, a pharmaceutically activeamount of a fusion protein or construct of the invention, and/or of apharmaceutical composition comprising the same.

The amino acid sequences, compounds, proteins, polypeptides, fusionproteins, or multivalent or multispecific constructs and/or thecompositions comprising the same are administered according to a regimeof treatment that is suitable for preventing and/or treating the diseaseor disorder to be prevented or treated. The clinician will generally beable to determine a suitable treatment regimen, depending on factorssuch as the disease or disorder to be prevented or treated, the severityof the disease to be treated and/or the severity of the symptomsthereof, the specific amino acid sequence, compound, protein,polypeptide, fusion protein, or multivalent or multispecific constructof the invention to be used, the specific route of administration andpharmaceutical formulation or composition to be used, the age, gender,weight, diet, general condition of the patient, and similar factors wellknown to the clinician.

Generally, the treatment regimen will comprise the administration of oneor more amino acid sequences, compounds, proteins, polypeptides, fusionproteins, or multivalent or multispecific constructs of the invention,or of one or more compositions comprising the same, in one or morepharmaceutically effective amounts or doses. The specific amount(s) ordoses to administered can be determined by the clinician, again based onthe factors cited above.

Generally, for the prevention and/or treatment of intended diseases anddisorders (i.e. those diseases and disorders which are usually treatedor prevented through the use of the therapeutic entity per se) anddepending on the specific disease or disorder to be treated, the potencyand/or the half-life of the specific amino acid sequences, compounds,proteins, polypeptides, fusion proteins, or multivalent or multispecificconstructs to be used, the specific route of administration and thespecific pharmaceutical formulation or composition used, the amino aminoacid sequences, compounds, proteins, polypeptides, fusion proteins, ormultivalent or multispecific constructs of the invention will generallybe administered in an amount between 1 gram and 0.01 microgram per kgbody weight per day, preferably between 0.1 gram and 0.1 microgram perkg body weight per day, such as about 1, 10, 100, 1000, or 2000microgram per kg body weight per day, either continuously (e.g. byinfusion), as a single daily dose or as multiple divided doses duringthe day. The clinician will generally be able to determine a suitabledaily dose, depending on the factors mentioned herein. It will also beclear that in specific cases, the clinician may choose to deviate fromthese amounts, for example on the basis of the factors cited above andhis expert judgment. Generally, some guidance on the amounts to beadministered can be obtained from the amounts usually administered forcomparable conventional antibodies or antibody fragments against thesame target administered via essentially the same route, taking intoaccount however differences in affinity/avidity, efficacy,biodistribution, half-life and similar factors well known to the skilledperson.

Usually, in the above method, a single amino acid sequence, compound,protein, polypeptide, fusion protein, or multivalent or multispecificconstruct of the invention will be used. It is however within the scopeof the invention to use two or more amino acid sequences, compounds,proteins, polypeptides, fusion proteins, or multivalent or multispecificconstructs of the invention in combination (e.g. as separatepreparations or combined in a single preparation).

The amino acid sequences, compounds, proteins, polypeptides, fusionproteins, or multivalent or multispecific constructs of the inventionmay also be used in combination with one or more furtherpharmaceutically active compounds or principles, i.e. as a combinedtreatment regimen, which may or may not lead to a synergistic effect.Again, the clinician will be able to select such further compounds orprinciples, as well as a suitable combined treatment regimen, based onthe factors cited above and his expert judgement.

In particular, the amino acid sequences, compounds, proteins,polypeptides, fusion proteins, or multivalent or multispecificconstructs of the invention may be used in combination with otherpharmaceutically active compounds or principles that are or can be usedfor the prevention and/or treatment of the diseases and disorders thatcan be prevented or treated with the amino acid sequences, compounds,proteins, polypeptides, fusion proteins, or multivalent or multispecificconstructs of the invention, and as a result of which a synergisticeffect may or may not be obtained.

The effectiveness of the treatment regimen used according to theinvention may be determined and/or followed in any manner known per sefor the disease or disorder involved, as will be clear to the clinician.The clinician will also be able, where appropriate and or a case-by-casebasis, to change or modify a particular treatment regimen, so as toachieve the desired therapeutic effect, to avoid, limit or reduceunwanted side-effects, and/or to achieve an appropriate balance betweenachieving the desired therapeutic effect on the one hand and avoiding,limiting or reducing undesired side effects on the other hand.

Generally, the treatment regimen will be followed until the desiredtherapeutic effect is achieved and/or for as long as the desiredtherapeutic effect is to be maintained. Again, this can be determined bythe clinician.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal, and more in particular a human being. As will beclear to the skilled person, the subject to be treated will inparticular be a person suffering from, or at risk from, the diseases anddisorders mentioned herein.

The invention will now be further illustrated by means of the followingnon-limiting Experimental Part and the non-limiting Figures, which show:

FIG. 1: alignment of the amino acid sequences of SEQ ID NO: 2 to 115(invention) and 17D12 (SEQ ID NO:1, reference);

FIG. 2: graph showing the results of the phage-ELISA assay described inExample 2.

FIG. 3: graph showing the results of the solution binding competitionassay described in Example 3.

FIGS. 4A and 4B: graphs showing the results of the alanine scanningexperiment described in Example 3.

FIG. 5: Graph showing results of surface plasmon resonance analysis ofthe binding of the Nanobody 2D3 (SEQ ID NO: 137), the Nanobody® fusionprotein of 2D3 and 17D12 (SEQ ID NO: 138, reference), and the Nanobody®fusion protein of 2D3 and 56H5 (SEQ ID NO: 139, invention) described inExample 6 to human serum albumin (HSA). Coating of the chip (CM5) wasperformed by amine coupling using NHS/EDC for activation andethanolamine for deactivation (Biacore amine coupling kit). Chip coatedwith ˜7000 RU human serum albumin (Sigma, 99% pure) and 2460 RUirrelevant protein antigen. 2D3 and 2D3-17D12 was successively injectedover the chip at concentrations of 1 μM and 5 μM. HBS-EP was used asflow buffer at a rate of 10 μl min-1. 20 μl of sample was injected for120 s. Note that the fusion protein of 2D3 and 17D12 (SEQ ID NO: 138) iscalled “2D3-56G4” in FIG. 5.

FIG. 6: Pharmacokinetic profile of cynomolgus monkeys administered withthe test item (Nanobody® construct 2D3-9GS-EXP56E4, SEQ ID NO: 142) andof cynomolgus monkeys administered with a negative control (Nanobody®2D3, SEQ ID NO:137).

FIGS. 7A to 7C are diagrams showing the results obtained in Example 9with the affinity matured versions of 56E4 described in Example 9 whenthese were tested in the phage competition assay described in Example 5;

FIGS. 8A and 8B are diagrams showing the results obtained in Example 10for the binding of the fusion proteins compounds 2D3-9GS-56E4-MycHis,2D3-9GS-59C2-MycHis, 2D3-9GS-59F2-MycHis and 2D3-9GS-59H12-MycHis tohuman serum albumin (FIG. 8A) and cynomolgus serum albumin (FIG. 8B).

FIG. 9: Mean (+/−SD; n=3) serum concentration-time profiles of2D3-9GS-EXP56E4, 2D3-9GS-EXP59C2, and 2D3 following i.v. bolusadministration at 2 mg/kg 2D3-9GS-EXP56E4, 2D3-9GS-EXP59C2 or 2D3,respectively in the male Cynomolgus monkey.

FIG. 10: Mean (+/−SD; n=3) plasma concentration-time profiles ofvWF-0053, vWF-0055, vWF-0056, and vWF0001 following i.v. bolusadministration at 2 mg/kg vWF-0053, vWF-0055, vWF-0056 (test items), andvWF0001 (control), respectively in the male Cynomolgus monkey.

FIG. 11: Mean (+/−SD; n=3) % RICO-time profiles following i.v. bolusadministration at 2 mg/kg vWF-0053, vWF-0055, vWF-0056 (test items), andvWF0001 (control), respectively in the male Cynomolgus monkey.

FIG. 12: diagram showing the results of the perfusion experimentsperformed in Example 15 with the anti vWF compounds of the inventionvWF-0053, vWF-0055 and vWF-0056.

FIG. 13: diagram showing the results obtained in the ELISA for theristocetin-induced binding to vWF performed in Example 15 with the antivWF compounds of the invention vWF-0053, vWF-0055 and vWF-0056.

EXPERIMENTAL PART Example 1 Examples of Amino Acid Sequences of theInvention

Some non-limiting examples of amino acid sequences of the invention aregiven as SEQ ID NO's 2 to 115 and 147 to 157 in Table II below. Analignment of the sequences of SEQ ID NO's 2 to 115 is given in FIG. 1.

Some preferred amino acid sequences of the invention are marked in boldtypeface underlined (see for example SEQ ID NO:12).

Of these, the amino acid sequences PMP56G11 (SEQ ID NO:68); PMP56E4 (SEQID NO: 14); PMP54H4 (SEQ ID NO: 106); PMP54H5 (SEQ ID NO: 33); PMP56H1(SEQ ID NO: 31); PMP56E2 (SEQ ID NO:47); PMP56G3 (SEQ ID NO: 35);PMP54G1 (SEQ ID NO:38); PMP56F1 (SEQ ID NO: 30); PMP54H2 (SEQ ID NO: 40)PMP56H9 (SEQ ID NO: 100); PMP56F2 (SEQ ID NO: 51); PMP26A3 (SEQ IDNO:26) and 01B3 (SEQ ID NO:115) are particularly preferredrepresentative examples of amino acid sequences of the invention.

The sequences of SEQ ID NO's: 147 to 157 are some preferred butnon-limiting examples of affinity matured variants (see Example 9 below)of one of the above sequences (in this case, of PMP56E4—SEQ ID NO:14)and thus are also some particularly preferred amino acid sequences ofthe invention. Of these, the sequences 59F2 (SEQ ID NO: 149); 59H12 (SEQID NO: 155) and 59C2 (SEQ ID NO: 156) are especially preferred.

The amino acid sequence called “17D12” (SEQ ID NO:1) is not an aminoacid sequence of the invention, but is a comparative amino acid sequencedescribed in the non-prepublished International applicationPCT/EP2007/063348.

All sequences of the invention below (SEQ ID NOs: 2 to 115 and 147 to157) are expected to be cross-reactive for both human serum albumin andcyno serum albumin The sequences SEQ ID NOs: 2 to 60 and 115 were testedfor binding to human serum albumin, and the sequences of SEQ ID NOs: 61to 114 were tested for binding to serum albumin from cynomolgus monkey.The sequences of SEQ ID NOs: 2-60 and 115 all bind better (as determinedusing the assays described in Examples 2 and/or 3) to human serumalbumin than the sequence of SEQ ID NO:1 (the same is expected for thesequences of SEQ ID NOs: 61 to 114). Data obtained for some of thesequences of SEQ ID NO's: 147 to 157 is presented in Examples 9 ff.

TABLE II Examples of amino acid sequences of the invention(SEQ ID NOs: 2-115 and 147-157). CLONE DESIGNATION SEQ ID NO:AMINO ACID SEQUENCE 17D12 SEQ ID NO: 1 AASYSDYDVFGGGTDFGP PMP56B2SEQ ID NO: 2 AARYFDYDVFGGGTPAGD PMP54D2 SEQ ID NO: 3 AARYFDYDVFGGGTDLGDPMP56E6 SEQ ID NO: 4 AARYYDYDVFGGGTPLGG PMP56F5 SEQ ID NO: 5AARYYDYDVFGGGTPLGG PMP56G6 SEQ ID NO: 6 AARYYDYDVFGGGTPLGG PMP56E3SEQ ID NO: 7 AARYYDYDVFGGGTPLGA PMP56C3 SEQ ID NO: 8 AARYYDYDVFGGGTPLGAPMP56E5 SEQ ID NO: 9 AARYYDYDVFGGGTPLGA PMP54B2 SEQ ID NO: 10AARYYDYDVFGGGTVVGE PMP54C1 SEQ ID NO: 11 AARYYDYDVFGGGTRSGE PMP56A6SEQ ID NO: 12 AARYYDYDVFGGGTAGGQ PMP56B4 SEQ ID NO: 13AARYWDYDVFGGGTPVGG PMP56E4 SEQ ID NO: 14 AARYWDYDVFGGGTPVGG PMP56B1SEQ ID NO: 15 AARYWDYDVFGGGTPQGE PMP56C2 SEQ ID NO: 16AARYWDYDVFGGGTPQGE PMP56G2 SEQ ID NO: 17 AARYWDYDVFGGGTDPGG PMP54D3SEQ ID NO: 18 AARYLDYDVFGGGTQLGS PMP54F3 SEQ ID NO: 19AARYLDYDVFGGGTDVGS PMP54C3 SEQ ID NO: 20 AARYLDYDVFGGGTPIGE PMP54C2SEQ ID NO: 21 AARYPDYDVFGGGTPVGG PMP56C6 SEQ ID NO: 22AARYPDYDVFGGGTPSGG PMP54E2 SEQ ID NO: 23 AALYRDYDVFAGGTPGGG PMP56B5SEQ ID NO: 24 AALYRDYDVFGGGTPVGG PMP56F6 SEQ ID NO: 25AALYRDYDVFGGGTPVGG PMP56A3 SEQ ID NO: 26 AALYDDYDVFGGGTPVGG PMP56D6SEQ ID NO: 27 AALYDDYDVFGGGTPVGG PMP56B3 SEQ ID NO: 28AAVYDDYDVFGGGTPVGG PMP56C5 SEQ ID NO: 29 AAMYYDYDVFGGGTPTGA PMP56F1SEQ ID NO: 30 AAWYTDYDVFGGGTPQGG PMP56H1 SEQ ID NO: 31AAWYRDYDVFGGGTPLGA PMP54B1 SEQ ID NO: 32 AAWYRDYDVFGGGTDEGS PMP56H5SEQ ID NO: 33 AAFYDDYDVFGGGTPAGG PMP56H3 SEQ ID NO: 34AAFYWDYDVFGGGTDPGA PMP56G3 SEQ ID NO: 35 AAFYWDYDVFGGGTDPGA PMP56G1SEQ ID NO: 36 AAYYFDYDVFGGGTPEGT PMP56C1 SEQ ID NO: 37AAYYFDYDVFGGGTPEGT PMP54G1 SEQ ID NO: 38 AATYFDYDVFGGGTAVGS PMP56G5SEQ ID NO: 39 AAAYLDYDVFGGGTPVGG PMP54H2 SEQ ID NO: 40AAAYWDYDVFGGGTSAGT PMP56B6 SEQ ID NO: 41 AAVYWDYDVFGGGTSLGD PMP56H6SEQ ID NO: 42 AAWYFDYDVFGGGTADGE PMP56F3 SEQ ID NO: 43AAWYFDYDVFGGGTADGE PMP54G3 SEQ ID NO: 44 AAYYYDYDVFGGGTPGGE PMP56A1SEQ ID NO: 45 AADYYDYDVFGGGTSVGE PMP56E1 SEQ ID NO: 46AAYYYDYDVFGGGTPGGE PMP56E2 SEQ ID NO: 47 AAYYYDYDVFGGGTPGGE PMP56A5SEQ ID NO: 48 AAYYRDYDVFGGGTPVGE PMP54G3 SEQ ID NO: 49AALYRDYDVFGGGTQVGE PMP56D4 SEQ ID NO: 50 AALYKDYDVFGGGTPGGE PMP56F2SEQ ID NO: 51 AAPYRDYDVFGGGTPRGE PMP56A2 SEQ ID NO: 52AAPYHDYDVFGGGTPVGE PMP54F2 SEQ ID NO: 53 AALYGDYDVFGGGTPLGE PMP54H1SEQ ID NO: 54 AASYLDYDVFGGGTPFGE PMP54E1 SEQ ID NO: 55AAFYRDYDVFGGGTGSGN PMP54G2 SEQ ID NO: 56 AAIYRDYDVFGGGTPLGQ PMP56D5SEQ ID NO: 57 AATYYDYDVFGGGTPLGQ PMP54H3 SEQ ID NO: 58AASYRDYDVFGGGTPRGW PMP54E3 SEQ ID NO: 59 AATYLDYDVFGGGTPDGR PMP56A4SEQ ID NO: 60 AAFYMDYDVFGGGTPRGQ PMP54G5 SEQ ID NO: 61AAPYFDYDVFGGGTARGG PMP54F5 SEQ ID NO: 62 AAPYFDYDVFGGGTEVGG PMP56A9SEQ ID NO: 63 AAPYFDYDVFGGGTPMGG PMP56B9 SEQ ID NO: 64AARYYDYDVFGGGTPGGV PMP56D7 SEQ ID NO: 65 AARYYDYDVFGGGTPGGV PMP56H10SEQ ID NO: 66 AARYYDYDVFGGGTSRGG PMP56G10 SEQ ID NO: 67VARYYDYDVFGGGTWSGD PMP56G11 SEQ ID NO: 68 AVRYYDYDVFGGGTSVGG PMP54G6SEQ ID NO: 69 AALYYDYDVFGGGTPEGI PMP56A10 SEQ ID NO: 70AALYYDYDVFGGGTAAGS PMP56A7 SEQ ID NO: 71 AALYYDYDVFGGGTPRGG PMP56C7SEQ ID NO: 72 AAYYYDYDVFGGGTALGG PMP56B11 SEQ ID NO: 73AADYYDYDVFGGGTVFGS PMP56D8 SEQ ID NO: 74 AATYYDYDVFGGGTSLGN PMP56G7SEQ ID NO: 75 AALYYDYDVFGGGTYKGS PMP54D6 SEQ ID NO: 76AATYYDYDVFGGGTDGGS PMP56C10 SEQ ID NO: 77 AARYWDYDVFGGGTPEGV PMP54B5SEQ ID NO: 78 AARYWDYDVFGGGTAQGE PMP54E6 SEQ ID NO: 79AARYWDYDVFGGGTPEGV PMP56A8 SEQ ID NO: 80 AARYWDYDVFGGGTPEGV PMP56B7SEQ ID NO: 81 AARYWDYDVFGGGTPEGV PMP56C9 SEQ ID NO: 82AARYWDYDVFGGGTPEGI PMP56D12 SEQ ID NO: 83 AARYWDYDVFGGGTPEGV PMP56E8SEQ ID NO: 84 AARYWDYDVFGGGTPEGV PMP56F10 SEQ ID NO: 85AGRYWDYDVFGGGTAQGA PMP56G9 SEQ ID NO: 86 AGRYWDYDVFGGGTAQ GA PMP56E11SEQ ID NO: 87 VAKYWDYDVFGGGTDSGG PMP56F7 SEQ ID NO: 88AASYWDYDVFGGGTPVGD PMP56B12 SEQ ID NO: 89 AAQYWDYDVFGGGTPKGE PMP54C6SEQ ID NO: 90 AALYRDYDVFGGGTPVGG PMP56A11 SEQ ID NO: 91AALYRDYDVFGGGTSAGV PMP56B10 SEQ ID NO: 92 AALYRDYDVFGGGTPSGV PMP56D11SEQ ID NO: 93 AALYRDYDVFGGGTPKGE PMP56D9 SEQ ID NO: 94AALYRDYDVFGGGTPKGE PMP56C8 SEQ ID NO: 95 AALYRDYDVFGGGTPSGV PMP56E9SEQ ID NO: 96 AALYRDYDVFGGGTPSGV PMP56F11 SEQ ID NO: 97AALYRDYDVFGGGTPRGG PMP56F9 SEQ ID NO: 98 AALYRDYDVFGGGTPKGE PMP56H7SEQ ID NO: 99 AALYRDYDVFGGGTPVGG PMP56H9 SEQ ID NO: 100AALYRDYDVFGGGTPRGS PMP56H11 SEQ ID NO: 101 AAFYRDYDVFGGGTPKGG PMP56Al2SEQ ID NO: 102 AAFYRDYDVFGGGTPKGG PMP54H5 SEQ ID NO: 103AAFYRDYDVFGGGTDMGN PMP54E5 SEQ ID NO: 104 AAWYRDYDVFGGGTPLGA PMP56D10SEQ ID NO: 105 AAWYRDYDVFGGGTPLGA PMP54H4 SEQ ID NO: 106AARYPDYDVFGGGTSMGQ PMP54B6 SEQ ID NO: 107 AAMYDDYDVFGGGTPSGA PMP54C5SEQ ID NO: 108 AAYYLDYDVFGGGTPGGG PMP54F6 SEQ ID NO: 109AAFYDDYDVFGGGTPAGG PMP54H6 SEQ ID NO: 110 AASYLDYDVFGGGTPGGG PMP56B8SEQ ID NO: 111 AAPYLDYDVFGGGTPEGS PMP56C12 SEQ ID NO: 112AALYSDYDVFGGGTPPGV PMP56E10 SEQ ID NO: 113 AAPYPDYDVFGGGTPQGS PMP56E12SEQ ID NO: 114 AAMYDDYDVFGGGTPSGA 01B3 SEQ ID NO: 115 AALYDDYDVFGGGTPAGG59A5 SEQ ID NO: 147 AARWWDYDVFGGGTPVGG 59C8 SEQ ID NO: 148AARYWDWDVFGGGTPVGG 59F2 SEQ ID NO: 149 AARYWDFDVFGGGTPVGG 59B3SEQ ID NO: 150 AARYWDFDAFGGGTPVGG 59B2 SEQ ID NO: 151 AARFWDYDVFGGGTPVGG60E6 SEQ ID NO: 152 AARYWDYDVFGGGTPVDG 60F1 SEQ ID NO: 153AARYWDYDVFGGGSQVGG 60G5 SEQ ID NO: 154 AARYWDYDVFGGGSPVGG 59H12SEQ ID NO: 155 AARSWDFDVFGGGTPVGG 59C2 SEQ ID NO: 156 AARDWDFDVFGGGTPVGG59H10 SEQ ID NO: 157 AARYWDFDVFGGGSPVGG

Example 2 Phage ELISA

5-fold serial dilutions of phage clones (starting from ˜5×10¹¹ phage)were added to 96-well Nunc Maxisorp plates coated with human serumalbumin (2 μg/ml in PBS, overnight at 4° C.; plates were blocked withSuperblock T20 (Pierce) for 1 h at room temperature). The microtiterplate was washed with wash buffer (PBS, 0.05% Tween 20) and bound phageswere detected with anti-M13 and goat-anti mouse IRDye conjugate(610-130-121, Rockland). The amount of IRDye bound was measured onOdyssey (LI-COR Biosciences). The dilution of phage was plotted againstmeasured near-infrared fluorescence intensity (FIG. 2). Clones 56 E2 and56 F2 show stronger binding to HSA compared to the amino acid sequenceAASYSDYDVFGGGTDFGP (SEQ ID NO:1).

Example 3 Solution Binding Competition ELISA

A competition ELISA was performed to determine the relative bindingaffinity for the selected phage clones. 96-well Nunc Maxisorp plateswere coated with 2 μg/ml HSA in coating buffer at 4° C. Plates wereblocked with SuperblockT20 (Pierce) for 1 h at room temperature. Themicrotiter plates were washed with wash buffer (PBS, 0.05% Tween 20).

Sixty μl of a 12.5 fold dilution of a 1012/ml phage stock was incubatedwith 60 μl of various concentrations of HSA (1.6-10000 nM finalconcentration) for 30 minutes at room temperature in a tissue culturemicrotiter plate. Unbound phage was captured by transferring 100 μl ofthe well mixture to the HSA coated Maxisorp plate and incubating at roomtemperature for 30 minutes. The plate with captured phage was washedwith PBS-0.05% Tween 20 at least five times. Bound phages were detectedwith anti-M13 and goat-anti mouse IRDye conjugate. The amount of IRDyebound was measured on Odyssey (LI-COR Biosciences). The % of phagebinding was calculated by the following equation: Phage binding%=fluorescence signal of well with competitor/fluorescence signal ofwell with no competitor* 100 (FIG. 3). The IC₅₀, the concentration ofHSA in solution that inhibits 50% of the phage binding, represents theaffinity.

When this assay is used to compare binding of an amino acid sequence ofthe invention to the amino acid sequence AASYSDYDVFGGGTDFGP (SEQ IDNO:1), the amino acid sequences of the invention bind “better” to therelevant serum albumin (e.g. to human serum albumin).

Example 4 Alanine Scanning of 17D12

Alanine scanning of the peptide 17D12 (SEQ ID NO:1) peptide wasperformed to identify amino acids within the peptide sequence amenablefor mutation to improve binding to HSA. The amino acid residues of the17D12-peptide were numbered from 1 to 18, as follows:

(SEQ ID NO: 1) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 A A  S  Y  S D Y D V F   G   G  G  T   D    F   G   P

Individual amino acids which were not already alanine in the originalsequence were mutated to alanine and effects on binding to HSA of eachvariant peptide were investigated. The 16 variant peptide constructs(with alanine substitutions at positions 3-18 in SEQ ID NO:1) weregenerated as N-terminal fusions with M13 bacteriophage geneIII and phagewere produced. Variant peptides expressed on phage were assayed forbinding to HSA. Binding was compared to binding of the wild-type peptidedisplayed on phage. A Maxisorp microtiter plate was coated with 2 μg/mLHSA and blocked with SuperBlock T20. Serial 2-fold dilutions of variantor wild-type phage in PBS+0.05% Tween-20+10% Superblock T20 (Pierce)were incubated for 1.5 h at room temperature. Bound phage were detectedusing anti-M13 (27-9420-01, GE Healthcare) and goat anti-mouse IRDye700(610-130-121, Rockland) antibodies and near-infrared fluorescenceintensity was measured on Odyssey (LI-COR Biosciences). For clarityreasons, the data are represented in two graphs (FIGS. 4A and 4B). Aminoacid substitutions in 17D12 that did not result in a significantdecrease in HSA binding were selected for randomization (underlined inthe sequence above).

For affinity maturation of 17D12, 6 residues were chosen forrandomization using an nnk codon (underlined in sequence), based onalanine scanning data and the functionality of the residues.

Example 5 Solution Binding Competition ELISA of Clones 01G7, 01B3 and01C7

The 3 clones listed in Table III below were tested in a solution bindingcompetition ELISA, as follows:

A competition ELISA was performed to determine the relative bindingaffinity for the selected phage clones. 96-well Nunc Maxisorp plateswere coated with 2 μg/ml HSA in coating buffer at 4° C. Plates wereblocked with SuperblockT20 (Pierce) for 1 h at room temperature. Themicrotiter plates were washed with wash buffer (PBS, 0.05% Tween 20).

45 μl phage stock was pre-incubated with 65 μl HSA solution 1.67 μM (1μM final) or 65 μl 16.9% Superblock T20 in PBS/0.05% Tween 20 for 30minutes at room temperature in a tissue culture microtiter plate.Unbound phage was captured by transferring 100 μl of the well mixture tothe HSA coated Maxisorp plate and incubating at room temperature for 30minutes. The plate with captured phage was washed with PBS-0.05% Tween20 five times. Bound phages were detected with anti-M13 and goat-antimouse IRDye conjugate. The amount of IRDye bound was measured on Odyssey(LI-COR Biosciences). The ratio of phage binding was calculated by thefollowing equation: fluorescence signal of well withcompetitor/fluorescence signal of well with no competitor (Table III).

TABLE III Solution binding competition assay Clone Ratio 1 μM/0 μM HSA01G7 (=56H5; SEQ ID NO: 33) 0.43 01B3 (SEQ ID NO: 115) 0.42 01C7 (=56A3;SEQ ID NO: 26) 0.47

Example 6 Construction of a Nanobody-Expedite Fusion Protein andAnalysis of Binding to HSA

HSA-binding peptides 17D12 (reference) and 56H5 (SEQ ID NO:33;invention) were each genetically fused at the C-terminus of the Nanobody2D3:

[SEQ ID NO: 137] EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSSvia the following linker sequence (that comprises a Gly4Ser-Gly3Serlinker and a flanking amino acid sequence GSA]

[SEQ ID NO: 140] GGGGSGGGSAand with the following C-terminal tag:

[SEQ ID NO: 141] AAAEQKLISEEDLNGAAHHHHHH.The resulting fusion proteins had the following sequences:

2D3-17D12 Fusion Protein:

[SEQ ID NO: 138] EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGSAAASYSDYDVFGGGTDFGPAAAEQKLISEEDLNGAAHHHHHH.

2D3-56H5 Fusion Protein:

[SEQ ID NO: 139] EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGSAAAFYDDYDVFGGGTPAGGAAAEQKLISEEDLNGAAHHHHHH.

The binding of the resulting 2D3-17D12 and 2D3-56H5 fusion proteins tohuman serum albumin was determined using surface plasmon resonanceanalysis. For this purpose, the fusion proteins were expressed in E.coli TG1 cells. The fusion proteins were purified by IMAC/SEC andbinding to HSA was assessed in BIAcore™ 3000, by injecting 1 μM and 5 μMof the 2D3-17D12 and 2D3-56H5 fusion proteins on a CM5 chip coated with˜7000 RU human serum albumin (Sigma, 99% pure) and 2460 RU an irrelevantprotein antigen (reference). Coating of the chip (CM5) was performed byamine coupling using NHS/EDC for activation and ethanolamine fordeactivation (Biacore amine coupling kit). HBS-EP was used as flowbuffer at a rate of 10 μl min-1. 20 μl of sample was injected for 120 s.The 2D3 Nanobody was injected as control.

FIG. 5 shows improved binding of the 2D3-56H5 fusion protein to HSAcompared to the 2D3-17D12 fusion protein (which is called 2D3-56G4 inFIG. 5), whereas, as expected, 2D3 does not bind at identicalconcentrations tested. Calculated affinity of the 2D3-56H5 fusionprotein for HSA is ˜1.2 μM (ka (1/Ms)=7.57E+03 and kd (1/s)=9.3E-03). Asa control, 5 μM of the fusion protein 2D3-56H5 was injected on CM5 chipcoated with high density of irrelevant protein (2400RU), but no specificbinding was detected.

Example 7 Pharmacokinetic Profile in Male Cynomolgus Monkeys

A Nanobody construct was prepared as a fusion of the peptide 56E4 (SEQID NO: 14, also referred to herein as PMP56E4) and the Nanobody 2D3 (SEQID NO:137), via a Gly4Ser-Gly3Ser (“9GS”) linker sequence.

The sequence of the Nanobody construct (referred to as 2D3-9GS-EXP56E4)used was:

[SEQ ID NO: 142] EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGSRYWDYDVFGGG TPVGG.

As a negative control, the Nanobody 2D3 was used (without the 56E4peptide).

The pharmacokinetic profile of this 2D3-9GS-EXP56E4 Nanobody construct(“construct” or “test item” hereafter) was analysed in male cynomolgusmonkeys of approximately 3 to 4 years old and was compared to the 2D3control (“control” or “negative control” hereafter). The construct andthe control were each injected in three monkeys. Both the construct andthe control were administered at a dose of 2 mg/kg via intravenousinfusion. Blood samples were taken at predose, 5 min, 20 min, 1 h, 2 h,4 h, 8 h, and 16 h after administration and at test days 2, 3, 5, 7, 9,12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57 afterthe start of the infusion. In order to obtain at least 0.25 mL serum peranimal per sampling time, a sufficient volume of whole blood waswithdrawn per sampling time and the serum was isolated after 1 h ofincubation at 37° C. The serum samples were stored at −80° C.

Serum samples were tested for serum levels of construct and the control,respectively, using the following ELISA assay.

96-well microtiter plates (Maxisorp, Nunc, Wiesbaden, Germany) werecoated for 1 hour at 37° C. with Recombinant Human ErbB2/Fc Chimera, CF(R&D Systems, Minneapolis) in PBS at 3 μg/mL for the negative controland 4.5 μg/mL for the test item. Wells were aspirated and blocked for 30minutes at room temperature (RT) with SuperBlock®T20 PBS (Pierce,Rockford, Ill.). After this blocking step, wells were washed withPBS-0.05% Tween20.

Preparations for the standards, QC samples and dilutions of the testsamples were performed in a non-coated (polypropylene) plate.

Standard Curve and QC-Samples:

Solutions at the required concentrations were prepared in PBS 0.1%casein and spiked into 100% monkey serum. To prepare standards and QCsamples, a 1/10 dilution of the pure monkey serum dilutions was made inPBS-0.1% casein.

Test Samples:

Dilution factors for the test samples were estimated, and varied from1/10 to 1/500. Samples were diluted 1/10 in PBS 0.1% casein in a firststep, and if needed, further dilution was done in PBS 0.1% caseincontaining 10% monkey serum. These sample dilutions were furtherserially diluted ⅕ in PBS 0.1% casein with 10% monkey serum over 2wells.

Standards, QC samples and the ⅕ dilutions of the test samples weretransferred onto the coated plate and incubated for 1 hour at RT.Afterwards the plates were washed and rabbit polyclonal anti-VHH K1,purified against protein A and Her2/Fc depleted, was added at 1 μg/mL inPBS 0.1% casein, and incubated for 1 hour at RT. After washing a 1/2000dilution in PBS 0.1% casein of horse radish peroxidase labelled goatanti-rabbit (Dakocytomation, Denmark) was added to the plate andincubated for 30 minutes at RT. This enzyme catalyzes a chemicalreaction with the substrate sTMB (3,3′,5,5′-tetramethylbenzidine, SDTreagents, Brussels, Belgium), which results in a colorimetric change.After stopping this reaction after 15 minutes using HCl (1N), theintensity of the colour was measured by a spectrophotometer, whichdetermines the optical density of the reaction product, using a 450 nmwavelength of light.

The concentration of the construct and the control in the serum sampleswas determined towards a standard curve of the construct and thecontrol, respectively. The concentration determination was performedusing the sigmoidal dose-response curve with variable slope. All serumsamples were tested minimally in duplicate. Average values werereported. For each sample standard deviations and precision between thedifferent results was calculated.

The PK profile is represented in FIG. 6.

The calculated terminal half-life of the Nanobodies® is summarized inTable IV.

TABLE IV Terminal half-life expressed in hours obtained in cynomolgusmonkeys after administration of the 2D3-9GS-EXP56E4 or the negativecontrol (2D3). Nanobody ® Terminal Half-life 2D3-9GS-EXP56E4 8.54 ± 0.79hr 2D3 (control) 2.04 ± 0.74 hrThe data obtained in the experiment described in this Example 7 is alsomentioned in Example 13 and FIG. 9.

Example 8 Crystal Structure of Peptide Based on EXP56E4 with Human SerumAlbumin and in Silico Modelling of the Interactions of this Peptide withHuman Serum Albumin

In order to determine the binding interaction and epitopes of thepeptides of the invention with human serum albumin, the crystalstructure of a co-crystal of the following peptide(AAARYWDYDVFGGGTPVGGAAA; SEQ ID NO:143) and human serum albumin wasdetermined, and also the interactions between the peptide of SEQ ID NO:143 and human serum albumin were modeled in silico. The peptide of SEQID NO:143 was based on the sequence of EXP56E4 (SEQ ID NO:14) and,compared to the sequence of EXP56E4, contains an additional N-terminalalanine residue and three C-terminal alanine-residues.

It should be noted that, compared to the amino acid sequence of SEQ IDNO:1, the amino acid sequence of SEQ ID NO: 143 contains one additionalN-terminal alanine residue. Thus, in the numbering used in this Example8, position 2 corresponds to position 1 of the sequence of SEQ ID NO: 1(see also Table II); position 3 corresponds to position 2 of thesequence of SEQ ID NO: 1, etc.

The crystal structure was determined as follows: the purified proteinswere used in crystallization trials employing both, a standard screen ofapproximately 1200 different conditions, as well as crystallizationconditions identified using literature data. Conditions initiallyobtained have been optimized using standard strategies, systematicallyvarying parameters critically influencing crystallization, such astemperature, protein concentration, drop ratio, etc. These conditionswere also refined by systematically varying pH or precipitantconcentrations. Crystals were obtained via the method ofco-crystallization.

Crystals have been flash-frozen and measured at a temperature of 100K.The X-ray diffraction have been collected at the SWISS LIGHT SOURCE(SLS, Villigen, Switzerland) using cryogenic conditions. Data wereprocessed using the programs XDS and XSCALE. The phase informationnecessary to determine and analyze the structure was obtained bymolecular replacement. Subsequent model building and refinement wasperformed with the software packages CCP4 and COOT. The peptideparameterization was carried out with the program CHEMSKETCH.

Modeling of the interaction was performed using ICM-Pro (Molsoft) andDiscovery Studio (Accelrys) with a force-field that is based on theparameters as described in Momany et al. (Momany et al. J. Phys. Chem.1975, 79, 2361-2381)

In respect of human serum albumin, for the purposes of this Example 8and the further disclosure herein, reference will be made to thesequence given under Genbank accession number AAA98797 (Minghetti etal., J. Biol. Chem. 261 (15), 6747-6757 (1986); SEQ ID NO: 144):

  1 mkwvtfisll flfssaysrg vfrrdahkse vahr F kdlge enfkalvlia faqylqqcpf 61 edhvklvnev tefaktcvad esaencdksl htlfgdklct vatlretyge madccakqep121 ernecflqhk dd NPNL p RL v  R pevdvmcta fhdneetflk k Y ly EI ar RH py F yapellf 181  F akrykaaft eccqaadkaa cllpkldelr de GK as Sakq rlkcaslqkf gerafkawav241 arlsqrfpka efaevsklvt dltkvhtecc hgdllecadd radlakyice nqdsissklk301 eccekpllek shciaevend empadlpsla adfveskdvc knyaeakdvf lgmflyeyar361 rhpdysvvll lrlaktyett lekccaaadp hecyakvfde fkplveepqn likqncelfe421 qlgeykfqna llvrytkkvp qvstptlvev srnlgkvgsk cckhpeakrm pcaedylsvv481 ln Q lcvlhek tpvsdrvtkc cteslvnrrp cfsalevdet yvpkefnaet ftfhadictl541 se K erqikkq talvelvkhk pkatkeqlka vmddfaafve kcckaddket cfaeegkklv601 aasqaalgl

Thus, when reference is made herein to a specific amino acid residue ofhuman serum albumin, the numbering of this amino acid residue will beaccording to the above sequence. It should however be noted that theabove sequence contains the following signal sequence(mkwvtfisllflfssaysrgvfn, SEQ ID NO:145). The sequence of mature humanserum albumin (without this signal sequence) is given below and in SEQID NO:146. This polypeptide was also used to determine the crystalstructure of the co-crystal with the peptide of SEQ ID NO: 143:

  1 dahksevahr  F kdlgeenfk alvliafaqy lqqcpfedhv klvnevtefa ktcvadesae 61 ncdkslhtlf gdklctvatl retygemadc cakqeperne cflqhkdd NP   NL p RL vR pev 121 dvmctafhdn eetflkk Y ly  EI ar RH py F y apellf Fakr ykaafteccq aadkaacllp 181 kldelrde GK  as Sakqrlkc aslqkfgera fkawavarls qrfpkaefae vsklvtdltk241 vhtecchgdl lecaddradl akyicenqds issklkecce kpllekshci aevendempa301 dlpslaadfv eskdvcknya eakdvflgmf lyeyarrhpd ysvvlllrla ktyettlekc361 caaadphecy akvfdefkpl veepqnlikq ncelfeqlge ykfqnallvr ytkkvpqvst421 ptlvevsrnl gkvgskcckh peakrmpcae dylsvvln Q l cvlhektpvs drvtkcctes481 lvnrrpcfsa levdetyvpk efnaetftfh adictlse K e rqikkqtalv elvkhkpkat541 keqlkavmdd faafvekcck addketcfae egkklvaasq aalgl

It should also be noted that Genbank accession number CAA00844 and EP0361991 give an alternative, synthetic amino acid sequence for humanserum albumin which—compared to the sequence of SEQ ID NO:144—containsone amino acid residue less than the sequence of AAA98797. Inparticular, in the sequence of CAA00844, and compared to the aminosequence of SEQ ID NO: 144, the amino acid residues KH on positions 463and 464 are replaced with a single amino acid residue N at position 463.Herein, when reference is made to the amino acid sequence of human serumalbumin and the amino acid residues present therein, reference is madeto the sequence and numbering given in SEQ ID NO:144].

From the crystal structure and modeling data, the following observationshave been made regarding the binding interaction of the peptide of SEQID NO: 143 and human serum albumin. It should be noted that theseobservations are given as exemplification only and do not limit theinvention to any specific (or complete) explanation or hypothesis onwhere (i.e. to which epitope) and how (i.e. via which amino acidresidues) the amino acid sequences of the invention bind to human serumalbumin However, it is assumed that the binding interactions andepitope(s) described below constitute one (preferred) way in which theamino acid sequences of the invention may bind to human serum albumin

-   -   The peptide of SEQ ID NO: 143 binds in a deep subpocket of        domain I of human serum albumin, and also has some interactions        with residues from domain III of human serum albumin    -   The peptide of SEQ ID NO: 143 may in particular bind to human        serum albumin via interaction with one or more of the following        amino acid residues: Asn (N) 133; Pro (P) 134; Asn (N) 135;        Leu (L) 136; Leu (L) 139; Arg (R) 141; Tyr (Y) 162; Glu (E) 165;        Ile (I) 166; His (H) 170; Phe (F) 173; Phe (F) 181; Gly (G) 213;        Lys (K) 214; Ser (S) 217; Gln (Q) 483; and/or Lys (K) 543. These        amino acid residues are indicated in UPPER CASE in the above        sequences.    -   In respect of the primary sequence of human serum albumin,        particularly important interactions appear to be the        interactions of the peptide of SEQ ID NO: 143 with the stretch        of amino acid residues that comprises the residues Asn (N) 133;        Pro (P) 134; Asn (N) 135; Leu (L) 136; Leu (L) 139 and Arg (R)        141; with the stretch of amino acid residues that comprises the        residues Tyr (Y) 162; Glu (E) 165; Ile (I) 166; His (H) 170;        Phe (F) 173; Phe (F) 181; and/or with the stretch of amino acid        residues that comprises the residues Gly (G) 213; Lys (K) 214        and Ser (S) 217;    -   In respect of the ternary structure of human serum albumin (as        deducted from the X-ray and modeling data), particularly        important interactions appear to be the interactions of the        peptide of SEQ ID NO: 143 with a rather hydrophobic subpocket        that is formed by (amongst others) residues the residues Leu (L)        139, Glu (E) 165, Ile (I) 166, His (H) 170, Phe (F) 173, Phe (F)        181, Gly (G) 213, Lys (K) 214, Ser (S) 217 and Gln (Q) 483 in        human serum albumin;    -   The three N-terminal alanine residues (Ala-1 to Ala-3) in the        peptide of SEQ ID NO: 143 could not be seen in the X-ray        structure. The results from in silico modeling results suggest        these alanine residues may be in contact with human serum        albumin    -   The Arg (R) residue at position 4 in peptide of SEQ ID NO: 143        likely forms a hydrogen bond with the amino acid residues        Asn (N) 133 & Asn (N) 135 of human serum albumin; and may also        forms electrostatic interactions with the main-chain oxygen        atoms of the Pro (P) 134 and Leu (L) 136 residues of human serum        albumin The Arg (R) residue at position 4 in peptide of SEQ ID        NO: 143 may also form an internal hydrogen bond with the Asp (D)        residue at position 7 of the peptide of SEQ ID NO: 143. The        crystal structure and modeling data suggests that this is likely        an important residue for the interaction between the peptide of        SEQ ID NO: 143 and human serum albumin;    -   The Tyr (Y) residue at position 5 in the peptide of SEQ ID NO:        143 likely forms a hydrogen-bond with the Lys (K) 543 residue of        human serum albumin via its main-chain. The crystal structure        and modeling data further suggests that the Tyr (Y) residue at        position 5 in peptide of SEQ ID NO: 143 is located in a        subpocket of HSA and is likely stabilized by the Trp (W) residue        at position 6 of peptide of SEQ ID NO: 143. The crystal        structure and modeling data also suggests that this is likely an        important residue for the interaction between the peptide of SEQ        ID NO: 143 and human serum albumin; and that the aromatic nature        of the Tyr residue at this position, although not strictly        needed at this position (the data suggests that other        hydrophobic residues may be present at this position), may        contribute to the stabilization with the Trp residue at position        6 of peptide of SEQ ID NO: 143;    -   The Trp (W) residue at position 6 in the peptide of SEQ ID NO:        143 appears to be nicely positioned between the Arg (R) 138 and        Lys (K) 543 residues of human serum albumin; and likely forms        strong electrostatic interactions with the Arg (R) 138 residue        of human serum albumin. It also appears that the aromatic nature        of the Trp (W) residue may be important for these interactions;        as well as for the stabilization with the Tyr (Y) residue at        position 5 in peptide of SEQ ID NO: 143. The crystal structure        and modeling data suggests that this is likely an important        residue for the interaction between the peptide of SEQ ID NO:        143 and human serum albumin. It should also be noted that in        serum albumin of cynomolgus monkey, mouse and rat, the amino        acid residue at position 138 is pro (P) instead of Arg (R); this        may lead to a reduced binding affinity of the amino acid        sequences of the invention for cyno, mouse and/or rat serum        albumin compared to human serum albumin;    -   The Asp (D) residue at position 7 in the peptide of SEQ ID NO:        143 likely forms an internal hydrogen bond with the Arg (R)        residue at position 4 in the peptide of SEQ ID NO: 143, and so        may be important for the local conformation of the peptide. The        crystal structure and modeling data also suggests that this        residue may potentially form a hydrogen bond with the His (H)        170 residue of human serum albumin, i.e. via its main-chain        oxygen atom (from the data, the Asp-7 side-chain does not appear        to have significant interactions with human serum albumin);    -   The Tyr (Y) residue at position 8 in the peptide of SEQ ID NO:        143 appears to bind in a hydrophobic subpocket that is formed by        the His (H) 170, Lys (K) 214, Ser (S) 217 and Gln (Q) 483        residues of human serum albumin: and may also have aromatic        interactions with the His (H) 170 residue of human serum albumin        (HSA) and/or internal aromatic interactions with the Phe (F)        residue at position 11 of the peptide of SEQ ID NO: 143. The        crystal structure and modeling data suggests that this is likely        an important residue for the interaction between the peptide of        SEQ ID NO: 143 and human serum albumin; but might possible be        replaced by another hydrophobic residue at position 8, as the        shape complementarity with the aforementioned hydrophobic        subpocket could possibly be further improved;    -   The Asp (D) residue at position 9 in the peptide of SEQ ID NO:        143 appears to undergo electrostatic interactions with the        Lys (K) 543 residue of human serum albumin; and also appears to        be partially solvent exposed. The crystal structure and modeling        data further suggests that this residue might possibly be        replaced by a Glu (E) residue at the same position, as such a        substitution might bring the carboxylic acid group closer to the        Lys (K) 543 in human serum albumin and so even further improve        these electrostatic interactions;    -   The Val (V) residue at position 10 in the peptide of SEQ ID NO:        143 appears to bind into a hydrophobic subpocket that is formed        by Leu (L) 139, Glu (E) 165, Ile (I) 166 and His (H) 170        residues of human serum albumin The crystal structure and        modeling data suggests that, due to the important hydrophobic        interactions and good shape complementarity with human serum        albumin, this is an important residue for the interaction        between the peptide of SEQ ID NO: 143 and human serum albumin;    -   The Phe (F) residue at position 11 in the peptide of SEQ ID NO:        143 appears to bind in a deep hydrophobic subpocket of human        serum albumin that is formed by the Ile (I) 166, His (H) 170,        Phe (F) 173, Phe (F) 181 & Gly (G) 213 residues of human serum        albumin Also, the main chain oxygen atom of the Phe (F) residue        at position 11 in the peptide of SEQ ID NO: 143 appears likely        to form a hydrogen-bond with the Tyr (Y) 162 residue in human        serum albumin The crystal structure and modeling data suggests        that this is likely an important residue for the interaction        between the peptide of SEQ ID NO: 143 and human serum albumin;    -   The three glycine residues at positions 12 to 14 of the peptide        of SEQ ID NO: 143 appear to bind deep into domain I of human        serum albumin and to make a turn which optimally fits with the        surface of human serum albumin The crystal structure and        modeling data suggests that this likely is important for the        interaction between the peptide of SEQ ID NO: 143 and human        serum albumin;    -   The Thr (T) residue at position 15 of the peptide of SEQ ID NO:        143 appears to form two main-chain hydrogen bonds with the        Leu (L) 139 and Arg (R) 141 residues. In addition, the Thr (T)        residue at position 15 of the peptide of SEQ ID NO: 143 appears        to form an internal hydrogen bond with the Asp (D) residue at        position 7 of the peptide of SEQ ID NO: 143; might possibly also        form a stabilizing internal hydrogen-bond with Asp (D) residue        at position 9 of the peptide of SEQ ID NO: 143.    -   The Pro (P) residue at position 16 of the peptide of SEQ ID NO:        143 might have a function in positioning of (and/or constraining        the optimal conformation for) the two hydrogen bonds of that are        formed by the Thr (T) residue at position 15 of the of peptide        of SEQ ID NO: 143    -   From the X-ray structure, no observations could be made for the        C-terminal part of the peptide of SEQ ID NO: 143 not seen in        X-ray structure. Modeling suggests that the C-terminal stretch        does not interact with HSA (except maybe for the Val (V) residue        at position 16, which is the residue from the C-terminal end        that is closest to human serum albumin compared to the other        C-terminal residues. The modelling data suggests the possibility        that the Val residue at position 16 could possibly be replaced        by a larger (d-) residue (such as, in particular, a Glu residue)        which could possibly interact with the Arg (R) 114 residue of        human serum albumin and in this way potentially also indirectly        contribute to the further stabilization of the Trp (W) residue        at position 6 (HSA) of the peptide of SEQ ID NO: 143.

Again, although the abovementioned X-ray and modeling data, as well asthe observations made based on that data, are non-limiting and given asexemplification only, it is assumed that other amino acid sequences withthe same or comparable amino acid sequences at positions correspondingto those mentioned above will undergo interactions with human serumalbumin that are essentially the same as and/or essentially similar tothe interactions described above for the peptide of SEQ ID NO: 143; andthat the abovementioned stretches of amino acid residues in the primarysequence of human serum albumin and/or the binding pockets on humanserum albumin described above form one or more important epitopes forthe binding of the amino acid sequences of the invention to human serumalbumin.

Example 9 Affinity Maturation of an Amino Acid Sequence of the Invention

In this example, 56E4 (AARYWDYDVFGGGTPVGG, SEQ ID NO:14) was chosen as astarting point for affinity maturation. 8 residues (bold/underlined)were chosen for randomization via parsimonious mutagenesis using acoding sequence for 56E4 but synthesized with a 70:10:10:10 mixture ofbases (70% original base and 10% of the other three bases), resulting ina frequency of 50% of the wild type amino at each randomized position.

The randomized peptide was expressed on the surface of M13bacteriophages as N-terminal fusion to geneIII protein using apUC19-derived phagemid vector. Four rounds of in solution selectionswere performed using biotinylated human serum albumin (HSA: A5763,Sigma), concentrations ranging from 1 μM to 1 nM. After incubation for 2h in presence of ovalbumin or casein as blocking agent, phages bound tobiotinylated HSA were captured on neutravidin and after washing thebound phages were eluted with 100 mM triethylamine and neutralized with1M Tris pH 7.5.

After respectively three and four selection rounds, monoclonal phagewere screened for binding on HSA, in the manner described in Example 2.Clones that bind to HSA (FIGS. 7A to 7C and Table V) were screened inphage competition ELISA, in the manner described in Example 5. The ratioof phage binding was calculated by the following equation: fluorescencesignal of well with 2 μM competitor/fluorescence signal of well with nocompetitor (Table VI).

TABLE V Alignment of clones resulting from affinitymaturation of 56E4 (shown at top) that bind to HSA Clone SEQ ID NO:sequence 59E4 SEQ ID NO: 14 AARYWDYDVFGGGTPVGG 59A5 SEQ ID NO: 147AARWWDYDVFGGGTPVGG 59C8 SEQ ID NO: 148 AARYWDWDVFGGGTPVGG 59F2SEQ ID NO: 149 AARYWDFDVFGGGTPVGG 59B3 SEQ ID NO: 150 AARYWDFDAFGGGTPVGG59B2 SEQ ID NO: 151 AARFWDYDVFGGGTPVGG 60E6 SEQ ID NO: 152AARYWDYDVFGGGTPVDG 60F1 SEQ ID NO: 153 AARYWDYDVFGGGSQVGG 60G5SEQ ID NO: 154 AARYWDYDVFGGGSPVGG 59H12 SEQ ID NO: 155AARSWDFDVFGGGTPVGG 59C2 SEQ ID NO: 156 AARDWDFDVFGGGTPVGG 59H10SEQ ID NO: 157 AARYWDFDVFGGGSPVGG

TABLE VI Solution binding competition assay for 56E4, 59C2, 59F2 and59H12 Clone Ratio 2 μM/0 μM HSA 56E4 0.82 59C2 0.91 59F2 0.69 59H12 0.87

Example 10 Construction of a Nanobody-Expedite Fusion Protein andAnalysis of Binding to HSA

HSA-binding peptides 56E4 (reference), 59C2, 59F2 and were eachgenetically fused at the C-terminus of the Nanobody 2D3 (SEQ ID NO: 137)using the linker sequence of SEQ ID NO: 140 (that comprises aGly4Ser-Gly3Ser linker and a flanking amino acid sequence GSA) and theC-terminal tag of SEQ ID NO:141. The resulting fusion proteins (forwhich the sequence are given below) were expressed in E. coli TG1 cellsand purified by IMAC/SEC, using standard vectors, conditions andtechniques.

2D3-9GS-56E4-MycHis:

[SEQ ID NO: 158] EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGSAAARYWDYDVFGGGTPVGGAAAEQKLISEEDLNGAAHHHHHH

2D3-9GS-59F2-MycHis:

[SEQ ID NO: 159] EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGSAAARYWDFDVFGGGTPVGGAAAEQKLISEEDLNGAAHHHHHH

2D3-9GS-59C2-MycHis:

[SEQ ID NO: 160] EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGSAAARDWDFDVFGGGTPVGGAAAEQKLISEEDLNGAAHHHHHH

2D3-9GS-59H12-MycHis:

[SEQ ID NO: 161] EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGSAAARSWDFDVFGGGTPVGGAAAEQKLISEEDLNGAAHHHHHH

The binding of the resulting 2D3-9GS-56E4-MycHis, 2D3-9GS-59C2-MycHis,2D3-9GS-59F2-MycHis and 2D3-9GS-59H12-MycHis fusion proteins to humanand cynomolgus serum albumin (HSA and CSA respectively) was determinedusing surface plasmon resonance analysis. For this purpose, binding toHSA and CSA was assessed in BIAcore™ 3000, by injecting concentrationseries of the fusion proteins ranging from 2 μM to 200 nM on a CM5 chipcoated with ˜3000 RU HSA or CSA. Coating of the chip (CM5) was performedby amine coupling using NHS/EDC for activation and ethanolamine fordeactivation (Biacore amine coupling kit). HBS-EP was used as flowbuffer at a rate of 4Sμ1 min-1. 90 μl of sample was injected for 120 s.

FIGS. 8A and 8B show improved binding of the 2D3-9GS-59C2-MycHis,2D3-9GS-59F2-MycHis and 2D3-9GS-59H12-MycHis fusion protein to HSA andCSA compared to 2D3-9GS-56E4-MycHis. Calculated affinities are shown inTable VII.

TABLE VII Kinetic values for MycHis tagged 2D3-Expedite fusionsCynomolgus monkey serum Human serum albumin albumin KD KD ka (1/ms) Kd(1/s) (nM) ka (1/ms) Kd (1/s) (nM) 2D3-9GS- 5.02E+03 4.97E−03 9914.01E+03 8.45E−03 2110 56E4- MycHis 2D3-9GS- 4.59E+03 3.04E−03 6633.45E+03 4.41E−03 1280 59H12- MycHis 2D3-9GS- 6.14E+03 3.51E−03 5714.85E+03 5.04E−03 1040 59F2- MycHis 2D3-9GS- 3.73E+03 1.79E−03 4812.75E+03 2.13E−03 775 59C2- MycHis

Example 11 Construction of Anti-HER2Nanobody-Expedite Fusion Proteinsfor PK Study

The HSA-binding peptide 59C2 without the two N-terminal alanine residues(RDWDFDVFGGGTPVGG; SEQ ID NO: 162) was genetically fused with aGly4Ser-Gly3Ser (9GS) linker sequence (GGGGSGGGS; SEQ ID NO:163) at theC-terminus of the anti-HER2Nanobody 2D3 (SEQ ID NO: 137). The fusionprotein was expressed and produced in essentially the same manner asdescribed in Example 12.

The resulting fusion protein had the following sequence:

2D3-9GS-59C2:

[SEQ ID NO: 164] EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGSRDWDFDVFGGG TPVGG

The binding of 2D3-9GS-59C2 to human, cynomolgus monkey and baboon serumalbumin was determined using surface plasmon resonance analysis andcompared with binding of and 2D3-9GS-56E4 (SEQ ID NO:142), essentiallyas described in Example 7. The binding was determined in BIAcore™ 3000,by injecting concentration series of the fusion proteins ranging from 2μM to 200 nM on a CM5 chip coated with ˜3000 RU HSA or CSA. Coating ofthe chip (CM5) was performed by amine coupling using NHS/EDC foractivation and ethanolamine for deactivation (Biacore amine couplingkit). HBS-EP was used as flow buffer at a rate of 4Sμ1 min-1. 90 μl ofsample was injected for 120 s. Calculated affinities are shown in TableVIII.

TABLE VIII Kinetic values for 2D3-9GS-56E4 and 2D3-9GS-59C2 Cynomolgusmonkey serum Human serum albumin albumin KD KD ka (1/ms) Kd (1/s) (nM)ka (1/ms) Kd (1/s) (nM) 2D3-9GS- 1.04E+04 4.97E−03 342 1.71E+04 8.47E−03495 56E4 2D3-9GS- 8.76E+03 1.94E−03 221 8.98E+03 2.47E−03 276 59C2

Example 12 Construction of Anti-VWF Nanobody-Expedite Fusion Proteinsfor PK/PD Study

The HSA-binding peptide 59C2 (without the two N-terminal alanineresidues; SEQ ID NO: 162) was genetically fused either as a monomer oras a dimer with a Gly4Ser-Gly3Ser (9GS) linker sequence between the twopeptides (RDWDFDVFGGGTPVGGGGGGSGGGSRDWDFDVFGGGTPVGG; SEQ ID NO: 166) tothe bivalent anti-VWF Nanobody 12A2_sv1-AAA-12A2_sv1 (vWF-001):

[SEQ ID NO: 165] DVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPESVEGRFTISRDNAKRTVYLQMNSLRAEDTAVYYCAAAGVRAEQGRVRTLPSEYTFWGQGTQVTVSSAAAEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPESVEGRFTISRDNAKRTVYLQMNSLRAEDTAVYYCAAAGVRAEQGRVR TLPSEYTFWGQGTQVTVSSeither at the C-terminus or interspaced between the two VHH buildingblocks via the Gly4Ser-Gly3Ser (9GS) linker.

For production, Escherichia coli strain TG1 containing the pAX102plasmids expressing the constructs were inoculated in 200 mL TBsupplemented with 50 mg/L kanamycin (Kan) and 2% glucose, and incubatedON at 30° C. and 200 rpm. For each construct 1×10 L bioreactorcontaining TBKan (50 mg/L)+2% glucose was inoculated with 1/50 of theobtained overnight pre-culture and further grown at 37° C. during thefollowing batch phase to obtain biomass. After 3 hours, the cultureswere induced with 1 mM IPTG and further grown at 30° C. for another 3hours during induction phase until OD600>10. The cultures were harvestedby centrifugation (Sigma 8K10 rotor; 7000 rpm; 20′; 4° C.), after whichthe clarified fermentation broth was stored at 4° C. and the cellpellets were stored at −20° C.

For purification, periplasmic extracts were prepared by re-suspendingthe pellets in 1 to 1.5 L peri-buffer (50 mM NaH2PO4, 300 mM NaCl pH8.0) and incubating for 40 minutes at 4° C. on a shaking platform at 200rpm. The suspensions were centrifuged at 7000 rpm for 40 minutes toclear the cell debris from the periplasmic extract, followed by afiltration step using a 0.45 μm filter. All the fusion proteins werecaptured via affinity chromatography using MabCapture A (Poros),followed by intermediate purification step via either CEX for thevWF-EXP molecules [Poros 50HS (Poros); equilibration buffer PBS, elutionbuffer PBS/1M NaCl, followed by binding and elution for vWF0056 onSource 15S; equilibration buffer 25 mM Hepes, 75 mM NaCl pH=8.0 andelution buffer: 25 mM Hepes, 175 mM NaCl pH=8.0] or via AEX for2D3-EXP59C2 [Poros 50HQ (Poros); equilibration buffer 25 mM Tris pH7.66,elution buffer 25 mM Tris pH7.79-500 mM NaCl]. Finally, all samples weretreated with OGP for LPS-removal, followed by a final size exclusionchromatography step using Superdex 75 pg (GE Healthcare) in D-PBS. TheOD280 nm was measured and the concentrations for the different fusionswere calculated. Samples were after sterile filtration stored at −20 C.LC/MS analysis indicated experimentally observed mass was in agreementwith the respectively theoretically expected masses of each construct.

The resulting fusion proteins had the following sequences:

12A2 sy1-9GS-59C2-9GS-12A2 sv1 (vWF-0053):

[SEQ ID NO: 167] DVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPESVEGRFTISRDNAKRTVYLQMNSLRAEDTAVYYCAAAGVRAEQGRVRTLPSEYTFWGQGTQVTVSSGGGGSGGGSRDWDFDVFGGGTPVGGGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPESVEGRFTISRDNAKRTVYLQMNSLRAEDTAVYYCAAAGVRAEQGRVRTLPSEYTFWGQGTQVTV SS12A2 sy1-9GS-59C2-9GS-59C2-9GS-12A2 sv1 (vWF-0054):

[SEQ ID NO: 168] DVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPESVEGRFTISRDNAKRTVYLQMNSLRAEDTAVYYCAAAGVRAEQGRVRTLPSEYTFWGQGTQVTVSSGGGGSGGGSRDWDFDVFGGGTPVGGGGGGSGGGSRDWDFDVFGGGTPVGGGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPESVEGRFTISRDNAKRTVYLQMNSLRAEDTAVYYCAAAGVRAEQGRVRTLPSEYTFWGQGTQVTVSS12A2 sv1-AAA-12A2 sy1-9GS-59C2 (vWF-0055):

[SEQ ID NO: 169] DVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPESVEGRFTISRDNAKRTVYLQMNSLRAEDTAVYYCAAAGVRAEQGRVRTLPSEYTFWGQGTQVTVSSAAAEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPESVEGRFTISRDNAKRTVYLQMNSLRAEDTAVYYCAAAGVRAEQGRVRTLPSEYTFWGQGTQVTVSSGGGGSGGGSRDWDFDVFGGGTPVGG12A2 sv1-AAA-12A2 sy1-9GS-59C2-9GS-59C2 (vWF-0056):

[SEQ ID NO: 170] DVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPESVEGRFTISRDNAKRTVYLQMNSLRAEDTAVYYCAAAGVRAEQGRVRTLPSEYTFWGQGTQVTVSSAAAEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPESVEGRFTISRDNAKRTVYLQMNSLRAEDTAVYYCAAAGVRAEQGRVRTLPSEYTFWGQGTQVTVSSGGGGSGGGSRDWDFDVFGGGTPVGGGGGG SGGGSRDWDFDVFGGGTPVGG

Example 13 Pharmacokinetic Profile in Male Cynomolgus Monkeys:Monovalent Nanobody

The Nanobody construct tested was a fusion of the peptide 59C2 (SEQ IDNO: 156) and the Nanobody 2D3 (SEQ ID NO:137). The sequence of thisconstruct (2D3-9GS-59C2) is given in Example 11 and SEQ ID NO: 164.

In this Example 13 and its corresponding FIG. 9, the data for anotherconstruct of the invention (2D3-9GS-56E4; SEQ ID NO:142) as obtained inExample 7 is also presented.

As a negative control, the Nanobody 2D3 was used.

For blood sampling and processing, the pharmacokinetic profile of theconstructs (2D3-9GS-EXP56E4 and 2D3-9GS-EXP59C2) and the negativecontrol 2D3 were determined in male Cynomolgus monkeys of approximately3 to 4 years old and was compared to that of the control (2D3). Theconstructs and the control were administered to three monkeys at a doseof 2 mg/kg by intravenous bolus injection. Blood samples 2D3 and for2D3-9GS-EXP56E4 were taken at predose, 5 min, 20 min, 1 h, 2 h, 4 h, 8h, and 16 h (test day 1), and on test days 2, 3, 5, 7, 9, 12, 15, 18,21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, and 57. While, for2D3-9GS-59C2 blood samples were taken only up to test day 33. In orderto obtain at least 0.25 mL serum per animal per sampling time, asufficient volume of whole blood was withdrawn per sampling time and theserum was isolated after 1 h of incubation at 37° C. The serum sampleswere stored at −80° C. until analysis.

For bioanalytical determination of the construct and the control articlein monkey serum, serum samples were tested for serum levels of theconstructs and control, respectively, using the following ELISA assay.96-well microtiter plates (Maxisorp, Nunc, Wiesbaden, Germany) werecoated for 1 hour at 37° C. with Recombinant Human ErbB2/Fc Chimera, CF(R&D Systems, Minneapolis) in PBS at 3 μg/mL for the negative controland 4.5 μg/mL for the test item. Wells were aspirated and blocked for 30minutes at room temperature (RT) with SuperBlock®T20 PBS (Pierce,Rockford, Ill.). After this blocking step, wells were washed withPBS-0.05% Tween20. Preparations for the standards, QC samples anddilutions of the test samples were performed in a non-coated(polypropylene) plate.

A standard curve and QC-samples were obtained as follows: solutions atthe required concentrations were prepared in PBS 0.1% casein and spikedinto 100% monkey serum. To prepare standards and QC samples, a 1/10dilution of the pure monkey serum dilutions was made in PBS-0.1% casein.

The dilution factors for the test samples were estimated, and variedfrom 1/10 to 1/500. Samples were diluted 1/10 in PBS 0.1% casein in afirst step, and if needed, further dilution was done in PBS 0.1% caseincontaining 10% monkey serum. These sample dilutions were furtherserially diluted ⅕ in PBS 0.1% casein with 10% monkey serum over 2wells.

Standards, QC samples and the ⅕ dilutions of the test samples weretransferred onto the coated plate and incubated for 1 hour at RT.Afterwards the plates were washed and rabbit polyclonal anti-VHH K1,purified against protein A and Her2/Fc depleted, was added at 1 μg/mL inPBS 0.1% casein, and incubated for 1 hour at RT. After washing a 1/2000dilution in PBS 0.1% casein of horse radish peroxidase labelled goatanti-rabbit (Dakocytomation, Denmark) was added to the plate andincubated for 30 minutes at RT. This enzyme catalyzes a chemicalreaction with the substrate sTMB (3,3′,5,5′-tetramethylbenzidine, SDTreagents, Brussels, Belgium), which results in a colorimetric change.After stopping this reaction after 15 minutes using HCl (1N), theintensity of the color was measured by a spectrophotometer, whichdetermines the optical density of the reaction product, using a 450 nmwavelength of light.

The concentration of the constructs and the control in the serum sampleswas determined towards a standard curve of the constructs and thecontrol, respectively. The concentration determination was performedusing the sigmoidal dose-response curve with variable slope. All serumsamples were tested minimally in duplicate. Average values werereported. For each sample standard deviations and precision between thedifferent results was calculated.

For the analysis of the pharmacokinetic data, descriptive statistics(mean and SD) were calculated per dose group and per sampling time pointusing Microsoft Excel 2007. In case all three values were BQL, BQL wasreported. When one or two out of three values were BQL, BQL values wereset to zero and the mean calculated. Individual serum concentration-timeprofiles were subjected to non-compartmental analysis (NCA) (Model 201;i.v. bolus injection) using WinNonlin Pro 5.1 (Pharsight Corporation,USA; 2006). The area under the curve (AUC) was estimated using the linup/log down rule. LLOQ values were treated as missing, except whencomprised between two values above the LLOQ, then they were set to zero.The concentration at time zero (C0) was estimated throughback-calculation based on the two first data points. The terminalelimination half-life (PM was calculated automatically (best-fit) usinga log-linear regression of the non-zero concentration-time data of thelog-linear portion of the terminal phase. A minimum of three points wereconsidered for the determination of λz.

The following main pharmacokinetic parameters were estimated: the serumconcentration at time zero (C0); the area under the serumconcentration-time curve extrapolated to infinity (AUCinf), total bodyclearance (CL), volume of distribution at steady-state (Vdss), and theterminal half-life (t½).

The results (for both the construct 2D3-9GS-59C2 as described in thisExample 13, as well as the construct 2D3-9GS-56E4 as described inExample 7) are shown in FIG. 9. In this FIG. 9, the mean serumconcentration time profile of 2D3-9GS-EXP56E4, 2D3-9GS-EXP59C2 and 2D3following an i.v. bolus administration at 2 mg/kg of 2D3-9GS-EXP56E4,2D3-9GS-EXP59C2 (test items) and 2D3 (control article), respectively inthe male Cynomolgus monkey is depicted.

In Table IX, the main pharmacokinetic parameters of 2D3-9GS-EXP56E4,2D3-9GS-EXP59C2, and 2D3 in the male Cynomolgus monkey are listed.

TABLE IX Main pharmacokinetic parameters (mean +/− SD; n = 3) of2D3-9GS-EXP56E4, 2D3-9GS-EXP59C2 (test items) and 2D3 (control article)following i.v. bolus administration of 2 mg/kg 2D3-9GS-EXP56E4,2D3-9GS-EXP59C2 or 2D3, respectively in the male Cynomolgus monkey.2D3-9GS- 2D3-9GS- EXP56E4 EXP59C2 2D3 Parameter Units Mean SD Mean SDMean SD C0 ug/ml 66.8 10.2 62.5 2.5 63.0 20.1 AUCinf ug * h/ml 458 171540 98 19.9 8.6 CL ml/h * kg 4.37 0.17 1.30 0.08 114 48 Vdss ml/kg 50.34.5 45.5 5.54 116 34 t½ h 8.54 0.79 20.9 4.23 2.04 0.74

Following an i.v. dose of 2 mg/kg, the C0-values were comparable for allthree compounds. However, the exposure and the corresponding total bodyclearance (CL) after administration of 2D3-9GS-EXP56E4 and2D3-9GS-EXP59C2 was respectively substantially higher and lower (onaverage 23-fold for 2D3-9GS-EXP56E4 and 77-fold for 2D3-9GS-EXP59C2,respectively) than after administration of the control article, 2D3.

The estimated values of the volume of distribution at steady-state(Vdss) were lower after administration of 2D3-9GS-EXP56E4 and2D3-9GS-EXP59C2, relative to 2D3.: 2D3-9GS-EXP56E4 and 2D3-9GS-EXP59C2had mean Vdss-values which were respectively 2.3- and 2.6-fold lowercompared to 2D3.

The terminal half-life (t½) was increased 4.2, and 10-fold from ca 2.0 hfor 2D3 to ca 8.5 h for 2D3-9GS-EXP56E4, and to about 21 h for2D3-9GS-EXP59C2, mainly as a result of the markedly decreased CL.

Example 14 Pharmacokinetic Profile in Male Cynomolgus Monkeys: BivalentNanobody Constructs

The bivalent Nanobody constructs tested were the constructs vWF 0053(SEQ ID NO:167); vWF 0055 (SEQ ID NO:169) and vWF-0056 (SEQ ID NO:170)described in Example 12. Of these, vWF-0056 (SEQ ID NO:170) has aC-terminal tag comprising two amino acid sequences of the inventionlinked via a linker (see SEQ ID NO: 166). The corresponding bivalentNanobody without any amino acid sequence of the invention (vWF-001, SEQID NO:165) was used as a reference.

The pharmacokinetic profile of the constructs was analyzed in malecynomolgus monkeys of approximately 3 to 4 years old and was compared tothe reference (SEQ ID NO:165). The constructs and the control were eachinjected in three monkeys at a dose of 2 mg/kg via an intravenous bolusinjection. Blood samples were taken at predose, 5 min, 20 min, 1 h, 2 h,4 h, 8 h (test day 1) after administration and at test days 2, 3, 5, 7,9, 12, 15, 18, 21, 24, 27, 30, 33. In order to obtain at least 1 mLplasma per animal per sampling time, a sufficient volume of whole bloodwas withdrawn per sampling time. Plasma was collected after whole bloodcentrifugation for 30 minutes at 2200 g at room temperature (RT). Theplasma samples were stored at −80° C. until analysis.

For bioanalytical determination of constructs and control article inmonkey plasma, plasma samples were tested for levels of constructs andthe control using ELISA based PK assays. The detection of control andconstructs in the ELISA assays is based on the binding of theseNanobodies with vWF and the assay set-ups are as such that totalvWF-binding Nanobody is detected.

For the reference (SEQ ID NO:165) and the construct of SEQ ID NO:167,96-well microtiter plates (Maxisorp, Nunc, Wiesbaden, Germany) werecoated overnight at 4° C. with neutravidin (Pierce) in 10:10 buffer at 3μg/ml. Wells were aspirated and blocked for 1 hour at RT with PBS/1%casein. After this blocking step, wells were washed with PBS/0.05%Tween20. A biotinylated bivalent Nanobody against the constructs wasadded to the neutravidin coated plate at 2 μg/ml in PBS/0.1% casein andincubated for 1 hour at RT. After the incubation step of this capturetool, wells were washed 3 times with PBS/0.05% Tween20.

Preparations of the standards, QC samples and dilutions of the testsamples were performed in a non-coated (polypropylene) plate. For thestandard curve and QC-samples, solutions at the required concentrationswere prepared in PBS/0.1% casein and spiked into 100% monkey plasma. Toprepare standards and QC samples, a 1/100 dilution of the pure monkeyplasma dilutions was made in IgM-Reducing Agent (ImmunochemistryTechnologies, Bloomington, USA) supplemented with 2,5% pooled humanplasma (referred to as sample diluent). For the test samples, dilutionfactors for the test samples were estimated, and ranged between 1/100 to1/9000 for the reference of SEQ ID NO:165 tests and between 1/100 and12000 for the construct of SEQ ID NO: 167. Samples were diluted 1/100 insample diluent in a first step, and if needed, further dilution was donein sample diluent supplemented with 1% cynomolgus plasma.

Standards, QC samples and diluted test samples were transferred onto thecoated plate and incubated for 1 hour at RT. Afterwards the plates werewashed followed by a complexation step with purified vWF (ZLB Behring).vWF diluted to 2.5 μg/ml for the assay for the reference of SEQ IDNO:165 and to 3 μg/ml for the assay for the construct of SEQ ID NO: 167in PBS/0.1% casein was incubated on the plates for 30 minutes at RT.Plates were washed and Nanobody/vWF complexes bound to the capture tooldetected with Rabbit anti-human vWF Ab (Dako, Denmark), diluted 1/2000in PBS/0.1% casein and incubated for 30 minutes at RT. After washing, a1/15000 dilution in PBS/0.1% casein of Horse-Radish-Peroxidase labelledgoat anti-rabbit Ab (Dako, Denmark) was added to the plate and incubatedfor 30 minutes at RT. The enzyme coupled to the Ab catalyzes a chemicalreaction with the substrate sTMB (3,3′,5,5′-tetramethylbenzidine, SDTreagents, Brussels, Belgium), resulting in a colorimetric change. Afterstopping this reaction after 10 minutes using HCl (1N), the intensity ofthe colour was measured using a spectrophotometer at 450 nm.

The concentrations of the reference of SEQ ID NO:165 or the construct ofSEQ ID NO: 167, respectively, in the plasma samples were determinedbased on the parameters of a 4-parameter logistic fit of the standardcurve (prepared using the reference of SEQ ID NO:165 or the construct ofSEQ ID NO: 167, respectively). All test samples were tested in 2independent runs and the reported values are the average of the 2analysis batches.

For the constructs of SEQ ID NO: 169 and 170, 96-well microtiter plates(Maxisorp, Nunc, Wiesbaden, Germany) were coated overnight at 4° C. witha monoclonal antibody (mAb) against the constructs at 6 μg/mL in PBS.Wells were aspirated and blocked for 1 hour at RT with PBS/1% casein.After this blocking step, wells were washed with PBS-0.05% Tween20.Preparations for the standards, QC samples and dilutions of the testsamples were performed in a non-coated (polypropylene) plate.

For the standard curve and QC-samples, solutions at the requiredconcentrations were prepared in PBS 0.1%/casein and spiked into 100%monkey plasma. To prepare standards and QC samples, a 1/100 dilution ofthe pure monkey plasma dilutions was made in IgM-reducing Agent(Immunochemistry Technologies, Bloomington, USA) supplemented with 2.5%pooled human plasma (referred to as sample diluent).

For the test samples, dilution factors for the test samples wereestimated, and ranged between 1/100 to 1/14000. Samples were diluted1/100 in sample diluent in a first step, and if needed, further dilutionwas done in sample diluent supplemented with 1% cynomolgus plasma.

Standards, QC samples and diluted test samples were transferred onto thecoated plate and incubated for 1 hour at RT. Afterwards the plates werewashed followed by a complexation step with purified vWF (ZLB Behring).vWF diluted to 3 μg/ml in PBS/0.1% casein was incubated on the platesfor 30 minutes at RT. Plates were washed and Nanobody/vWF complexesbound to the capture tool detected with a Rabbit anti-human vWF Ab(Dako, Denmark), diluted 1/2000 in PBS/0.1% casein and incubated for 30minutes at RT. After washing, a 1/15000 dilution in PBS/0.1% casein ofHorse-Radish-Peroxidase labelled goat anti-rabbit Ab (Dako, Denmark) wasadded to the plate and incubated for 30 minutes at RT. The enzymecoupled to the Ab catalyzes a chemical reaction with the substrate sTMB(3,3′,5,5′-tetramethylbenzidine, SDT reagents, Brussels, Belgium),diluted ½ with TMB weakener, SDT reagents) which results in acolorimetric change. After stopping this reaction after 20 minutes usingHCl (1N), the intensity of the colour was measured using aspectrophotometer, which determines the optical density of the reactionproduct, at 450 nm.

The concentrations of each of the constructs of SEQ ID NO: 169 and 170in the plasma samples were determined based on the parameters of a4-parameter logistic fit of the standard curve (prepared using therelevant construct). All test samples were tested in 2 independent runsand the reported values are the average of the 2 runs.

For pharmacokinetic data analysis, descriptive statistics (mean and SD)were calculated per dose group and per sampling time point usingMicrosoft Excel 2007. In case all three values were BQL, BQL wasreported. When one or two out of three values were BQL, BQL values wereset to zero and the mean calculated. Individual plasmaconcentration-time profiles were subjected to non-compartmental analysis(NCA) (Model 201; i.v. bolus injection) using WinNonlin Pro 5.1(Pharsight Corporation, USA; 2006). The area under the curve (AUC) wasestimated using the lin up/log down rule. LLOQ values were treated asmissing, except when comprised between two values above the LLOQ, thenthey were set to zero. The concentration at time zero (C0) was estimatedthrough back-calculation based on the two first data points. Theterminal elimination half-life (t½) was calculated automatically(best-fit) using a log-linear regression of the non-zeroconcentration-time data of the log-linear portion of the terminal phase.A minimum of three points were considered for the determination of λz.

The following main pharmacokinetic parameters were estimated: the plasmaconcentration at time zero (C0); the area under the plasmaconcentration-time curve extrapolated to infinity (AUCinf), total bodyclearance (CL), volume of distribution at steady-state (Vdss), and thedominant half-life (t_(1/2), dominant), and the terminal half-life (t½).

In FIG. 10, the mean plasma concentration time profiles of theconstructs of SEQ ID NO: 167 (vWF-0053), SEQ ID NO: 169 (vWF-0055), SEQID NO: 170 (vWF-0056) and the reference (vWF0001) following an i.v.bolus administration at 2 mg/kg of vWF-0053, vWF-0055, and vWF-0056(test items) and vWF-0001 (control article), respectively in the maleCynomolgus monkey are presented.

After i.v. injection, the plasma levels of the control article,vWF-0001, dropped rapidly during the first two hours afteradministration from about 45 ug/ml to ca 2 ug/ml. This initial drop islikely the result of rapid elimination of unbound vWF-0001 by thekidneys. Beyond 2 h post-dose, a slower decline in plasma levels wasobserved, which is most likely explained by the slower elimination ofthe vWF0001-vWF complex by the liver.

In the temporal plasma concentration profiles of the various constructs,no such rapid initial decline in plasma levels was apparent. This islikely related to binding of the constructs to monkey albumin,preventing rapid clearance through the kidneys.

In Table X, the main pharmacokinetic parameters of the variousconstructs and the control vWF0001 in the male Cynomolgus monkey arelisted.

TABLE X Main pharmacokinetic parameters (mean +/− SD; n = 3) ofvWF-0053, vWF-0055, and vWF-0056 and vWF0001 following i.v. bolusadministration of 2 mg/kg vWF-0053, vWF-0055 and vWF-0056 (test items)or vWF0001 (control article), respectively in the male Cynomolgusmonkey. vWF-0053 vWF-0055 Parameter Units Mean SD Mean SD C0 ug/ml 48.93.1 46.5 9.1 AUCinf ug * h/ml 1320 88 1590 148 CL ml/h * kg 1.52 0.101.26 0.12 Vdss ml/kg 60.0 4.6 63.3 2.6 t½ h 33.2 0.30 40.5 3.9 dominantt½ h 31.8 2.2 27.1 2.2 terminal vWF-0056 vWF-0001 Parameter Units MeanSD Mean SD C0 ug/ml 69.4 16.2 45.3 1.3 AUCinf ug * h/ml 4140 607 62.611.0 CL ml/h * kg 0.489 0.067 32.7 5.7 Vdss ml/kg 38.5 3.1 727 51 t½ h75.5 7.6 0.477 0.057 dominant t½ h 30.4 7.9 22.9 3.6 terminal

Relative to control, the calculated total body clearance (CL) of theconstructs was substantially lower. The mean CL of vWF-0053, vWF-0055,and vWF-0056 was decreased 22-, 26-, and 67-fold, respectively comparedto the control.

The effect on the Vdss was less pronounced: vWF-0053, vWF-0055, andvWF-0056 had a Vdss-values which were on average 12-, 11-, and 19-foldlower relative to vWF0001.

Relative to vWF0001, the dominant half-life (t½ dominant) was markedlyincreased 70-, 85-, 158-fold, respectively for vWF-0053, vWF-0055, andvWF-0056. The terminal half-life (t½ terminal), which likely reflectselimination of the construct-vWF remained essentially the same (seeTable 1).

To evaluate the chemical stability of vWF0055, the Nanobody® vWF0055 wasstored at 37° C. After 1, 2 and 4 weeks a sample was taken and analyzedfor chemical or proteolytic modifications via RP-HPLC (Zorbax 300SB-C3,4.6×150 mm (5 μm); trifluoroacetic acid/acetonitrile). These analysesshowed that after 4 weeks incubation at 37° C. in D-PBS, neitherchemical modifications nor proteolytic degradation occurred (data notshown).

Example 15 Pharmacodynamic Profile and Activity Assays

For the constructs used in Example 14, pharmacodynamic characteristicsupon compound administration were measured by means of a ristocetincofactor activity assay (Biopool). The ristocetin cofactor activity is afunctional assay for VWF, measuring the capacity of VWF to interact withthe platelet receptor glycoprotein Ib using ristocetin as a modulator.

For pharmacodynamic data analysis, descriptive statistics (mean and SD)were calculated per dose group and per sampling time point usingMicrosoft Excel 2007. Response parameters and associated statistics forthe overall time course were calculated by noncompartmental analysis ofthe response-time data using WinNonlin Pro 5.1 (Pharsight Corporation,USA; 2006). The non-compartmental analysis was based on a model forpharmacodynamic data (Model 220). The threshold value was set at 20%based on extensive historical PK/PD data on the vWF-0001 compound.Preclinical studies have shown that a complete inhibition of thepharmacodynamic marker are correlated with full antithrombotic efficacy.The following main pharmacodynamic parameters were determined: timebelow the threshold (Time below T), area under the threshold (AUC belowT), time at which the % RICO first drops below the threshold(T_(onset)), and time at which the % RICO first returns back above thethreshold (T_(offset)).

In FIG. 11, the temporal time profiles of the % RICO measurementsfollowing an i.v. bolus administration at 2 mg/kg of vWF-0053, vWF-0055,and vWF-0056 (test items) and vWF-0001 (control article), respectivelyin the male Cynomolgus monkey are shown.

In Table XI, the main pharmacodynamic parameters of the variousconstructs and the control vWF-0001 in the male Cynomolgus monkey aftera single i.v. dose at 2 mg/kg are presented.

TABLE XI Main pharmacodynamic parameters (mean +/− SD; n = 3) ofvWF-0053, vWF-0055 and vWF-0056 and vWF0001 following i.v. bolusadministration of 2 mg/kg vWF-0053, vWF-0055 and vWF-0056 (test items)or vWF0001 (control article), respectively in the male Cynomolgusmonkey. vWF-0053 vWF-0055 Parameter Units Mean SD Mean SD AUC (% RICO *h) 379 291 781 406 below T Time (h) 39.9 11.0 127 26 below T Tonset (h)0.079 0.003 0.074 0.006 Toffset (h) 40.0 11.0 127 26 vWF-0056 vWF-0001Parameter Units Mean SD Mean SD AUC (% RICO * h) 1709 945 6.60 4.35below T Time (h) 202 6 1.21 0.1 below T Tonset (h) 0.079 0.003 0.0770.002 Toffset (h) 202 6 1.28 0.135

After i.v. injection, a rapid and comparable onset of action wasobserved (as evaluated by the % RICO measurements) following i.v.application of both the control article (vWF0001) and the variousconstructs (vWF-0053, vWF-0055 and vWF-0056). The T_(onset) values wereestimated at ca 5 minutes. Compared to control, T_(offset) and hence thetime below the threshold (Time below T), and also the AUC under thethreshold (AUC below T) were markedly increased after i.v.administration of the constructs.

The time below the threshold after i.v. bolus administration of vWF0001was on average 1.21 h and had increased 33-, 105-, and 167-fold afterapplication of the respective constructs (vWF-0053, vWF-0055, andvWF-0056).

The mean AUC under the threshold after i.v. bolus administration was6.60% RICO*h after vWF0001 administration; its value was increased 57-,118-, and 259-fold after dosing with vWF-0053, vWF-0055, and vWF-0056,respectively.

The activity of the constructs was determined/confirmed in perfusionexperiments (see for example Example 16 of WO 04/062551 or Example 4 ofWO 06/122825) and a standard inhibition ELISA for measuring inhibitionof ristocetin-induced binding of VWF to platelets.

The perfusion experiments were performed with a single-pass perfusionchamber under non-pulsatile flow conditions using a modified smallperfusion chamber with a slit height of 0.1 mm and a slit width of 2 mm.Thermanox coverslips (Nunc, Rochester, N.Y.) were coated overnight with0.5 mg/mL Horm collagen type III (Nycomed) and subsequently blocked withHepes buffer containing 1% human serum albumin Citrated human blood waspreincubated at 37° C. for 5 mM with or without addition of testcompound, and then perfused through the chamber for 5 mM at a wall shearrate of 1600 s⁻¹ using an infusion/withdrawal pump (pump 22, model2400-004, Harvard Apparatus, Holliston, Mass.). After the perfusion run,the coverslips were rinsed in Hepes buffered saline (10 mM Hepes, 150 mMNaCl, pH 7.4) and platelets were fixed with 5% glutaraldehyde andstained with May-Grünwald and Giemsa. Platelet deposition of thecoverslip was evaluated as platelet surface coverage of 10 randomlychosen pictures using light microscopy (Olympus BX61 microscope usingAnalysis Five digital imaging solutions analysis software).

These results are shown in FIG. 12, and demonstrate that VWF0055,VWF0056 and their non half life extended equivalents dose-dependentlyand completely inhibit platelet adhesion to fibrillar collagen atarterial shear rate. Effective concentration was similar for VWF0055 andVWF0056 compared to VWF0001.

For determining the inhibition of ristocetin-induced binding of VWF toPlatelets, microtiter plates (Maxisorp, Nunc) were coated overnight at4° C. with 0.1 mg/mL poly-L-Lysine (Sigma, St Louis, Mo.) in PBS. After3 times washing with phosphate buffered saline (PBS), wells wereincubated for 1 hour at room temperature (RT) with formalin fixed humanplatelets (Dade Behring, Newark, Del.) which were diluted two-fold inPBS or—as a blank—with PBS. Wells were washed 3 times with PBS andblocked for 2 hours at RT with PBS containing 4% bovine serum albumin(BSA, Sigma). A dilution series of compound was prepared in human plasmaand was preincubated for 30 min at RT with 1.5 mg/mL ristocetin (abp,NJ, USA) after which the mixture was transferred to the coated wells.After 1.5 hours incubation at 37° C., wells were washed 6 times with PBSand residual bound vWF was detected for 1 hour at RT with a 1/2000dilution of anti-VWF polyclonal antibodies labeled with horse radishperoxidase (Dako, Glostrup, Denmark). Visualization was obtained withesTMB (SDT reagents, Germany) and the coloring reaction was stopped with1M hydrochloric acid after which the absorbance was determined at 450nm. For the analysis of the data, the absorbance values were correctedusing the absorbance of the respective blanks.

The results are shown in FIG. 13 and demonstrate that all compoundsdose-dependently and completely block the ristocetin-induced binding ofVWF to the platelet surface. Similar potency was observed for theexpedite constructs compared to the non-half life extended parent.Potency of compounds with 2 VWF binding domains was much higher comparedto the potency of a mock variant, in which one of the VWF bindingdomains was exchanged by an irrelevant Nanobody®. This suggests avidbinding of the bivalent compounds to the multimeric VWF and henceconfirms functionality of the 2 VWF binding units.

Example 16 Biacore Analysis to Determine pH Dependency of the Binding ofvWF0055 to HSA and cynoSA

The pH dependent binding of vWF0055 on HSA and cynoSA was investigatedby surface plasmon resonance using a Biacore 3000 instrument byassessing the affinity at three different pH's. In brief, HSA and cynoSAwere amine-coupled to a separate CM5 sensor chip at a density ofrespectively 1800RU and 1900RU. Diluted samples, ranging inconcentration between 25 nM to 1 μM vWF0055 were prepared in threebuffers. The three buffers used contained 50 mM phosphate, 150 mM NaCland 0.005% surfactant P and were adjusted to either pH5, pH7 or pH8.Samples were injected at a flow-rate of 45 μL/min, association anddissociation were monitored during 120 sec and 300 sec. Binding curveswere subsequently used to calculate the K_(D), association—anddissociation-rate constants with the BiaEvaluation software. The highestaffinity was observed at pH 7, both for HSA and cynoSA. At pH5 theaffinity of vWF0055 was decreased about 10-fold.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

All references disclosed herein are incorporated by reference, inparticular for the teaching that is referenced hereinabove.

1. An isolated amino acid sequence that specifically binds human serumalbumin and that comprises: (i) an Arg (R) residue that forms a hydrogenbond with the amino acid residues Asn (N) 133 & Asn (N) 135 of humanserum albumin and/or forms electrostatic interactions with themain-chain oxygen atoms of the Pro (P) 134 and Leu (L) 136 residues ofhuman serum albumin; and (ii) a Trp (W) residue that forms electrostaticinteractions with the Arg (R) 138 residue of human serum albumin.
 2. Theisolated amino acid sequence according to claim 1, that comprises: (i) asequence motif RXWD, in which R is arginine, X may be any amino acid, Wis tryptophan, and D is aspartic acid; and/or (ii) a sequence motif GGG,in which G is glycine.
 3. The isolated amino acid sequence according toclaim 2, wherein in the sequence motif RXWD, X is tryptophan (W),tyrosine (Y), phenylalanine (F), serine (S) or aspartic acid (D).
 4. Theisolated amino acid sequence according to claim 2, wherein the aminoacid sequence comprises sequence motif RXWD and GGG.
 5. The isolatedamino acid sequence according to claim 2, that comprises: (i) thesequence motif RXWD, in which R is arginine, X may be any amino acid, Wis tryptophan, and D is aspartic acid; and/or (ii) a sequence motif FGGG(SEQ ID NO:120), in which F is phenylalanine and G is glycine.
 6. Theisolated amino acid sequence according to claim 5, wherein in thesequence motif RXWD, X is tryptophan (W), tyrosine (Y), phenylalanine(F), serine (S) or aspartic acid (D).
 7. The isolated amino acidsequence according to claim 5, wherein the amino acid sequence comprisessequence motif RXWD and FGGG (SEQ ID NO:120).
 8. The isolated amino acidsequence according to claim 5, that comprises (i) the sequence motifRXWD, in which R is arginine, X may be any amino acid, W is tryptophan,and D is aspartic acid; and/or (ii) a sequence motif DVFGGG (SEQ IDNO:129), in which D is aspartic acid, V is valine, F is phenylalanineand G is glycine.
 9. The isolated amino acid sequence according to claim8, wherein in the sequence motif RXWD, X is tryptophan (W), tyrosine(Y), phenylalanine (F), serine (S) or aspartic acid (D).
 10. Theisolated amino acid sequence according to claim 8, wherein the aminoacid sequence comprises sequence motif RXWD and DVFGGG (SEQ ID NO:129).11. The isolated amino acid sequence according to claim 1 that: a) hasno more than 9 amino acid differences over the full length of theisolated amino acid sequence with the amino acid sequenceAASYSDYDVFGGGTDFGP (SEQ ID NO:1); and that: b) binds better to humanserum albumin than the amino acid sequence AASYSDYDVFGGGTDFGP (SEQ IDNO:1).
 12. The isolated amino acid sequence according to claim 11, inwhich, compared to the amino acid sequence of SEQ ID NO.1: the serineresidue (S) at position 3 of SEQ ID NO:1 is replaced by an amino acidresidue chosen from arginine (R), proline (P), phenylalanine (F),tyrosine (Y), tryptophan (W), histidine (H), leucine (L), isoleucine(I), valine (V) or methionine (M); and/or the serine residue (S) atposition 5 of SEQ ID NO:1 is replaced by an amino acid residue chosenfrom arginine (R), proline (P), phenylalanine (F), tyrosine (Y),tryptophan (W) or histidine (H); and/or the aspartate residue (D) atposition 15 of SEQ ID NO:1 is replaced by an amino acid residue chosenfrom proline (P), alanine (A), glycine (G), serine (S) or threonine (T);and/or the phenylalanine residue (F) at position 16 of SEQ ID NO:1 isreplaced by proline (P), leucine (L), isoleucine (I), valine (V),methionine (M), alanine (A), glycine (G), serine (S) or threonine (T);and/or the proline residue (P) at position 18 of SEQ ID NO:1 ismaintained or replaced by aspartic acid (D), glutamic acid (E),glutamine (Q), asparagine (N), alanine (A), glycine (G), serine (S) orthreonine (T); and which amino acid sequence optionally comprises one ormore further suitable amino acid insertions, deletions and/orsubstitutions.
 13. The isolated amino acid sequence according to claim11, that comprises: (i) a sequence motif RXWD, in which R is arginine, Xmay be any amino acid, W is tryptophan, and D is aspartic acid; and/or(ii) a sequence motif GGG in which G is glycine.
 14. The isolated aminoacid sequence according to claim 5, wherein in the sequence motif RXWD,X is tryptophan (W), tyrosine (Y), phenylalanine (F), serine (S) oraspartic acid (D).
 15. The isolated amino acid sequence according toclaim 5, wherein the amino acid sequence comprises sequence motif RXWDand GGG.
 16. The isolated amino acid sequence according to claim 13,that comprises: (i) the sequence motif RXWD, in which R is arginine, Xmay be any amino acid, W is tryptophan, and D is aspartic acid; and/or(ii) a sequence motif FGGG (SEQ ID NO:120) in which F is phenylalanineand G is glycine.
 17. The isolated amino acid sequence according toclaim 16, wherein in the sequence motif RXWD, X is tryptophan (W),tyrosine (Y), phenylalanine (F), serine (S) or aspartic acid (D). 18.The isolated amino acid sequence according to claim 16, wherein theamino acid sequence comprises sequence motif RXWD and FGGG (SEQ IDNO:120).
 19. The isolated amino acid sequence according to claim 16,that comprises (i) the sequence motif RXWD, in which R is arginine, Xmay be any amino acid, W is tryptophan, and D is aspartic acid; and/or(ii) a sequence motif DVFGGG (SEQ ID NO:129) in which D is asparticacid, V is valine, F is phenylalanine and G is glycine.
 20. The isolatedamino acid sequence according to claim 19, wherein in the sequence motifRXWD, X is tryptophan (W), tyrosine (Y), phenylalanine (F), serine (S)or aspartic acid (D).
 21. The isolated amino acid sequence according toclaim 19, wherein the amino acid sequence comprises sequence motif RXWDand DVFGGG (SEQ ID NO:129).
 22. The isolated amino acid sequenceaccording to claim 1, wherein said amino acid sequence is such that,when it is linked or fused to a therapeutic moiety, compound, protein orother therapeutic entity, the compound thus obtained has a longerhalf-life than a corresponding compound or construct in which saidtherapeutic moiety, compound, protein or other therapeutic entity islinked or fused to the amino acid sequence of SEQ ID NO:1; andpreferably has a half-life that is the same or longer than acorresponding compound or construct in which said therapeutic moiety,compound, protein or other therapeutic entity is linked or fused to theamino acid sequence of SEQ ID NO:14.
 23. The isolated amino acidsequence according to claim 1, that is cross-reactive with serum albuminfrom cynomolgus monkeys (Macaca fascicularis).
 24. A compound orconstruct which comprises at least one amino acid sequence according toclaim 1 and at least one therapeutic moiety.
 25. A compound or constructwhich comprises at least two amino acid sequences according to claim 1and at least one therapeutic moiety.
 26. A compound or construct whichcomprises at least one tandem repeat comprising at least two amino acidsequences according to claim 1 and at least one therapeutic moiety.